DMD Fast Forward. Published on February 14, 2011 as doi:10...

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Pharmacokinetic interaction of the antiparasitic agents ivermectin and spinosad in dogs

Stewart T. Dunn, Laura Hedges, 1Kathleen E. Sampson, 2Yurong Lai, Sean Mahabir, 2Larissa

Balogh, and Charles W. Locuson

Pfizer Animal Health, Veterinary Medicine Research & Development, Metabolism & Safety,

333 Portage Street, KZO-300-421NW, Kalamazoo, MI 49001

Pfizer Animal Health, Metabolism & Safety (STD, LH, and CWL)

Pfizer Animal Health, Biometrics (SM)

Pfizer Global Research & Development, Pharmacokinetics, Dynamics, and Metabolism,

Chesterfield, MO (KES, YL, and LB)

DMD Fast Forward. Published on February 14, 2011 as doi:10.1124/dmd.110.034827

Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on February 14, 2011 as DOI: 10.1124/dmd.110.034827

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a) Running title: Interaction of ivermectin and spinosad in dogs

b) Corresponding author:

Charles W Locuson

Pfizer Animal Health, Veterinary Medicine Research and Development, Metabolism & Safety,


T: 269-833-2505

F: 269-833-7721

[email protected]

c) Text pages : 20

Tables 2

Figures 2

References 42

Words in Abstract 242

Words in Introduction 684

Words in Discussion 1591

d) Non-standard abbreviations: area-under-the-curve, AUC; central nervous system, CNS; blood-

brain barrier, BBB; N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-

2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine, CP100356; maximum plasma drug concentration

observed, Cmax,obs; drug-drug interaction, DDI; gamma aminobutyric acid, GABA; liquid

chromatography tandem mass spectrometry, LS-MS/MS


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Abstract

Neurological side effects consistent with ivermectin toxicity have been observed in dogs when

high doses of the common heartworm prevention agent ivermectin are coadministered with

spinosad, an oral flea prevention agent. Based on numerous reports implicating the role of the

ATP binding cassette drug transporter P-glycoprotein (P-gp) in ivermectin efflux in dogs, an in

vivo study was conducted to determine whether ivermectin toxicity results from a

pharmacokinetic interaction with spinosad. Beagle dogs were randomized to three groups

treated orally, in parallel: Treatment Group 1 (T01) received ivermectin (60 μg/kg), Treatment

Group 2 (T02) received spinosad (30 mg/kg), and Treatment Group 3 (T03) received both

ivermectin and spinosad. While spinosad pharmacokinetics were unchanged in the presence of

ivermectin, ivermectin plasma pharmacokinetics revealed a statistically significant increase in

AUC (3.6-fold over the control) when coadministered with spinosad. The majority of the

interaction is proposed to result from inhibition of intestinal and/or hepatic P-gp-mediated

secretory pathways of ivermectin. Furthermore, in vitro transwell experiments with a human

MDR1-transfected MDCKII cell line showed polarized efflux at concentrations ≤2 µM,

indicating that spinosad is a high affinity substrate of P-gp. In addition, spinosad was a strong

inhibitor of the P-gp transport of digoxin, calcein AM (IC50 = 3.2 µM), and ivermectin (IC50 =

2.3 µM). The findings suggest spinosad, acting as a P-gp inhibitor, increases the risk of

ivermectin neurotoxicity by inhibiting secretion of ivermectin to increase systemic drug levels,

and by inhibiting P-gp at the blood-brain barrier.


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Introduction

Ivermectin is one of the most effective and widely used antiparasitic agents ever discovered

because of its broad spectrum activity against numerous endo- and ectoparasites, especially

nematodes and arthropods (Geary, 2005; Omura, 2008). Chemically, ivermectin is a high

molecular weight natural product macrocyclic lactone produced by the actinomycete,

Streptomyces avermitilis (Campbell et al., 1983). Originally developed for veterinary use,

ivermectin is commonly used to eliminate gastrointestinal nematodes in livestock and

heartworms in companion animals. In several instances ivermectin has also proven to be an

effective treatment for worm infections, as well as mites, lice, and scabies in human medicine

(Omura, 2008). A number of companies currently market ivermectin and have adapted a range

of formulations for dosing by the oral, parenteral, and topical routes.

Much of ivermectin’s success could be attributed to its high therapeutic index. For instance, in

dogs, the effective heartworm prevention dose is 6 µg/kg once-monthly, but the more difficult to

treat dermatological Demodex infections are often treated daily with extra-label ivermectin doses

in excess of 50-fold the heartworm prevention dose (Mueller, 2004). The primary factors

contributing to the therapeutic index of ivermectin appear to be: (1) the high affinity of

ivermectin for its primary pharmacological targets in parasites, the glutamate-gated chloride ion

channels (Yates et al., 2003; Wolstenholme and Rogers, 2005); (2) the absence of the glutamate-

gated chloride (anion) channels in mammalian hosts (Raymond and Sattelle, 2002); and (3)

“protection” from ivermectin binding to its secondary target, γ-aminobutyric acid (GABA)-gated

chloride channels, because the expression of these GABA channels in mammals is mostly

limited to the central nervous system (CNS) (Campbell et al., 1983). When the limits of the


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safety margin are exceeded in the treatment of difficult to treat infections, the toxicological

syndromes appear to be consistent with GABA receptor modulation caused by ivermectin-

stimulated neuronal GABA release (Lovell, 1990).

Despite the well-deserved superdrug status held by ivermectin, drug interaction concerns have

recently arisen in dogs for use of extra-label doses in conjunction with the oral flea preventative,

spinosad. According to a warning statement issued by the Food and Drug Administration,

increased incidence of ivermectin toxicoses have been observed during co-administration of the

two antiparasitic agents in dogs

(http://www.fda.gov/AnimalVeterinary/NewsEvents/CVMUpdates/ucm047942.htm). The

authors are not aware of any study detailing the rate of occurrence of adverse events with extra-

label ivermectin doses. When administered with ivermectin at its heartworm prevention dose,

the safety of spinosad has never been questioned based on field experience. Recently, the safety

of extra-label doses of the anthelmintic milbemycin oxime has also been confirmed with

concurrent spinosad treatment (Sherman et al., 2010).

Interestingly, a clue to the potential mechanism behind the ivermectin-spinosad drug interaction

in dogs comes from a well-characterized P-glycoprotein (P-gp) mutation in dogs found

predominantly in the collie breed, but also other breeds (Mealey et al., 2001; Mealey and Meurs,

2008). Ivermectin is a substrate and inhibitor of the transmembrane drug transporter P-gp

(Didier and Loor, 1996). A four base pair deletion in the ABCB1 gene encoding P-gp results in

a truncated, non-functional protein (Mealey et al., 2001). This P-gp variant is associated with

increased sensitivity to ivermectin due to the enhancement of BBB drug penetration (Pulliam et


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al., 1985; Mealey et al., 2001). Clearly, clinician knowledge of the mutation is relevant during

ivermectin treatment, which is associated with neurotoxicity at high doses (≥100 µg/kg) typically

tolerated by wild-type allele carriers (Hopkins et al., 1990; Mealey, 2004; Fecht and Distl, 2008).

Because the safe use of high dose ivermectin therapies is related to P-gp function, it has been

conjectured that spinosad may inhibit ivermectin efflux by P-gp in dogs with wild-type P-gp

alleles. In such an instance, ivermectin would be predicted to have greater central nervous

system (CNS) penetration and GABA-related pharmacology. Since ivermectin is a P-gp

substrate that is primarily eliminated in feces/bile and not extensively metabolized, it is also

reasonable to envision how P-gp inhibition could increase circulating ivermectin via decreased

hepatic or intestinal secretion. The goal of this work was to test whether the ivermectin-spinosad

interaction in dogs is related to P-gp inhibition via pharmacokinetic studies and in vitro P-gp

efflux and inhibition experiments.


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Methods

Chemicals and Reagents. Dosing material was commercially available ivermectin solution

(Ivomec® (1%) in sterile solution 40% glycerol formal and propylene glycol, q.s. ad 100%.

Spinosad tablets (Comfortis™) were obtained from commercial retail resources for research use.

Ivermectin was purchased from Sigma-Aldrich (St. Louis, MO). An internal standard for

ivermectin, 23-hydroxy-doramectin, was prepared by Pfizer Inc. (Groton, USA). Spinosyn A

was purchased from Chembiotek Research International Pvt. Ltd (Worcestershire, UK), and an

internal standard consisting of spinosad containing a substituted amine was prepared by Pfizer

Inc (Sandwich, UK). Methanol and acetonitrile were obtained from Honeywell Burdick and

Jackson (Muskegon, MI). N-methylimidazole, trifluoroacetic anhydride, and human albumin

were obtained from Sigma-Aldrich (St Louis, MO). Acetic acid was obtained from Mallinckrodt

Laboratory Chemicals (Phillipsburg, NJ). Formic acid was obtained from EMD Chemicals Inc.

(Darmstadt, Germany). The solid phase extraction cartridges (C18 Isolute, 2 ml capacity with

100 mg separating medium) were obtained from Biotage (Charlottesville, VA). Minimum

Essential Medium-α (MEMα), fetal bovine serum (FBS), 10 mM MEM non-essential amino

acids, 10,000 units/ml penicillin-10,000 μg/ml streptomycin, 0.25% Trypsin-EDTA, Dulbecco’s

Phosphate Buffered Saline (D-PBS) and Hank’s Balanced Salt Solution (HBSS), pH 7.4, and

customized HBSS containing 25 mM glucose and 20 mM Hepes, pH 7.4 (transport buffer), were

purchased from Invitrogen Corp. (Carlsbad, CA). Vybrant™ Multidrug Resistance Assay Kit

and calcein-acetoxymethyl ester (calcein AM) in anhydrous DMSO were purchased from

Molecular Probes (Eugene, OR). The calcein AM was aliquoted into single use vials and stored

at –20oC under nitrogen gas in a dessicator.


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Study animals and treatment group randomization. All procedures in this study were conducted

in compliance with Animal Welfare Act Regulations. The use of research animals was

performed under the guide of an Institutional Animal Care and Use Committee approved Animal

Use Protocol. Study subjects were purpose bred beagle dogs ranging in age from 3-7 years.

Animals were allocated to treatments and runs by weight according to a randomized block design

with one-way treatment structure. Blocking was based on run location within the study room.

The experimental unit for treatment was the animal. Nine dogs were assigned to one of three

groups for oral dosing with either ivermectin (T01), spinosad (T02), or concurrent doses of

spinosad and ivermectin (T03).

Dosing and blood draws. Ivermectin (69 µg/kg, average) and spinosad (>30 mg/kg) were

administered orally via gavage and by chewable tablet, respectively. The dose of ivermectin was

10-fold higher than used for routine heartworm prevention to enable pharmacokinetic analysis.

Spinosad was administered at the recommended dose using the dose banding guidelines

provided. Each dog was fed 30 min prior to dosing to stimulate digestive processes. After

dosing, all subjects were administered 15 mL of water by syringe to ensure delivery of dose. An

intensive blood sampling regimen followed the dose with samples taken at pre-dose, and 1, 2, 3,

4, 6, 10, 24, 48, and 72 h, and 5, 7, 10, and 14 d. Two mL blood samples were collected via

jugular venipuncture with vacutainer tubes containing K2-EDTA anticoagulant. Plasma was

prepared by centrifuging and stored at -20 °C until analysis. Clinical signs were recorded at 2, 4,

and 10 h, and once daily for the remainder of the study. No adverse events were observed in any

study subjects.


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Plasma analysis. Ivermectin in plasma was assayed by liquid chromatography with fluorescence

detection after solid phase extraction and chemical derivatization. Standards and quality control

samples (prepared from separate weighings) were prepared by the addition of appropriate

volumes of standard solutions of ivermectin to aliquots of control dog plasma. Internal standard

solution (75:25 water:acetonitrile) was added to each standard and sample, mixed, and then

centrifuged. An Isolute extraction plate was primed with 2 mL methanol per well and allowed to

drain under gravity. Wells were then washed with 2 mL water. Plasma standards and samples

were then applied to the extraction plate, which was then washed with 2 mL water, followed by 1

mL 75:25 water:methanol, and dried under vacuum. The extraction plate was then eluted with 2

mL methanol. The eluate was collected in a 96-well plate (2 mL capacity), and evaporated to

dryness under a stream of nitrogen gas at 60 oC using a sample concentrator (TurboVap 96,

Caliper Life Sciences, Hopkinton, MA). N-methyl imidazole (60 μL of 50 % v/v in acetonitrile)

was added to each well in the plate, followed by trifluoroacetic anhydride (100 μL of 50 % v/v in

acetonitrile) to derivatize the samples. Detection was accomplished using a Waters Acquity

ultra-performance liquid chromatography system and fluorescence detector (Waters Corp.,

Milford, MA) with an excitation wavelength of 365 nm, and an emission wavelength of 475 nm.

Chromatographic separations were performed using an Acquity BEH C18 column, 1.7 um, 2.1 x

100 mm, and gradient elution. The flow rate was 500 uL per min, with a column temperature of

45 ºC and an injection volume of 10 μL. Solvent A was water and solvent B was 0.2 % acetic

acid in water, methanol, and acetonitrile (4:32:64 % v/v). Gradient conditions began at 50 % B,

increasing to 99 % B over 3.5 min, holding at 99% B for 3.5 min, then returning to initial

conditions over 2.5 min. Ivermectin eluted at 6.08 min, and internal standard at 5.45 min.


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Standard curves were shown to be linear in dog plasma over the concentration range covering the

unknown samples and no weighting was applied to the fit (r = 0.9967).

Spinosyn A was assayed by liquid chromatography separation and tandem mass spectrometry

(LC-MS/MS) methods. Standards, quality controls, and samples were prepared using the

Hamilton MicroLab Star (Reno, NV). Appropriate volumes of standard solutions of spinosyn A

were added to aliquots of control dog plasma in a 96-well format. Similarly, appropriate

volumes of control solvent were added to sample plasma, to maintain a common matrix. Internal

standard was then added to each standard and sample. The plate was thoroughly vortex mixed

and centrifuged and aliquots of supernatant transferred to an injection plate. Mass spectrometry

detection of test compounds was accomplished with an Applied Biosystems MDS/Sciex API

4000 triple quadrupole mass spectrometer (Foster City, CA). Chromatographic separations were

performed using a Zorbax Eclipse XDB C8 column, 3.5 um, 2.1 x 50 mm, and gradient elution.

The flow rate was 600 µL per min, with an injection volume of 10 μL. Solvent A was 0.1 %

formic acid and solvent B was methanol. Gradient conditions began at 10 % B, increasing to 90

% B over 1 min, holding at 90% B for 1.4 min, then returning to initial conditions over 0.5 min.

Spinosad eluted at 1.89 min, and internal standard at 2.06 min. Analysis was performed in

positive ion mode at unit resolution for the spinosad forosamine transition of 732.5 → 142.3.

Calibration curves were shown to be quadratic up to 1000 ng/mL in dog plasma. 1/X weighting

was used to provide the fit (r = 0.9982).

Pharmacokinetic analysis. Pharmacokinetic calculations were performed using the

noncompartmental approach (linear trapezoidal rule for AUC calculation) with the aid of Watson

(v7.2, Innaphase, Inc; Wayne, PA). The ivermectin and spinosad plasma concentrations used to


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calculate the terminal elimination rate constant and half-life were selected from the 72-336 h

time points.

Statistical analysis. Plasma concentrations for ivermectin and spinosad from the nine study

animals were analyzed using linear mixed models for repeated measures. Separate repeated

measures models were used for each drug. The models contained fixed effects for treatment, time

and the treatment by time interaction, and random effects for block, the block by treatment

interaction, and residual. Plasma concentrations were logarithmically transformed prior to

statistical analysis. If the treatment main effect or treatment by time interaction was significant,

pair-wise comparisons between treatment groups were performed. Treatment comparisons (two-

tailed) within time points were conducted at the 10% level of significance to compensate for the

small number of subjects in each treatment group.

The pharmacokinetic parameters were analyzed using mixed linear models with fixed effects for

treatment and random effects for block and residual. Pharmacokinetic parameters, with the

exception of Tmax,obs, were logarithmically transformed prior to statistical analysis. If the

treatment main effect was significant pair-wise comparisons between treatment groups were

performed. Treatment comparisons (two-tailed) were conducted at the 10% level of significance.

All statistical analyses were performed by using SAS 9.2 (SAS Institute, Cary, NC).

Cell culture. A MDCK II single cell isolation was double-sorted by flow cytometry for low P-gp

efflux function against calcein-AM accumulation, then expanded in culture for use in

transmonolayer assays (manuscript in preparation, L. Di, Pfizer Global Research &


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Development, Groton, CT). This subpopulation is referred to as MDCK Low Efflux, or

MDCKLE and was used to quantify passive permeability for comparison to efflux studies with

MDCK-MDR1. Cell stocks were grown in culture for not longer than 4 months, and P-gp

expression level and function was checked regularly to ensure the lack of function is maintained

over time. MDCKLE and human MDR1 gene transfected MDCK II cells (MDCK-MDR1; from

Dr. P. Borst, The Netherlands Cancer Institute, Amsterdam, The Netherlands), were cultured in

MEMα with L-glutamine containing 10% FBS, 100 µM MEM non-essential amino acids, 100

units/ml penicillin and 100 µg/ml streptomycin. Cells were maintained at 37ºC in humidified

incubator with 5% CO2, with culture media refreshed at 2-3 day intervals.

P-gp transwell efflux assay. Cells were plated at 6 x 104/well onto 24 well insert plates (BD

Biosciences, Bedford, MA) with a 1 µm pore polyethylene terephthalate filter and grown for 4

days. On day of study, the confluent monolayers were rinsed twice and then pre-incubated for

30-40 min with HBSS transport buffer, pH 7.4. Assays were performed using triplicate wells for

each direction of transport, either apical to basolateral (A→B) or basolateral to apical (B→A).

Compounds were diluted to the indicated concentrations in transport buffer and applied to donor

chamber. Human albumin (0.3 % v/v) had to be added to the buffer in donor and receiver

compartments in order to characterize the transport of ivermectin. The positive control substrate

digoxin was included in the studies to confirm transporter function. Transporter inhibitors were

added to both apical and basolateral chambers. Plates were incubated at 37οC for 2 h. Samples

were removed from donor chambers at t=0 min, and from both donor and receiver chambers at

t=120 min. Samples were diluted to 50% acetonitrile containing internal standard, and analyte

concentrations were determined by LC/MS/MS.


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The apparent permeability (Papp, cm/sec) was determined for both A→B and B→A directions by

the following calculation:

in which A = area of filter membrane, CD(0) = initial concentration of the test drug, dMr = the

amount of transported drug and dt = time elapsed. The efflux ratio (ER) was calculated from

(Papp, B→A)/(Papp, A→B). The ER in MDCKLE cells is used to define a null-P-gp transcellular flux.

A ratio of ratios (ER MDCK-MDR1 / ER MDCK-LE) greater than 1.7 has been established as the cutoff

to identify P-gp substrates (Feng et al., 2008).

Ivermectin efflux inhibition. Ivermectin (1 µM) was added to transwell MDCK-MDR1 plates

and the Papp determined for the B→A direction as described above. Experiments were performed

in the presence of spinosad (0.1 – 30 µM) with each concentration being tested in triplicate. The

inhibition of Papp for ivermectin was normalized to the Papp value obtained in the absence of

inhibitor and the resulting data fit to a one-site relative IC50 inhibition model using GraphPad

Prism software v5.02 (GraphPad Software, La Jolla, CA):

slope Hill)50(log

min,max,min,,

101

)(×−+

−+=

xIC

appappappobservedapp

PPPP

Calcein AM accumulation assay. MDCK-MDR1 cells were seeded into 96-well clear bottom

black wall sterile culture plates (BD Biosciences, Bedford, MA) at 5.3 x 103 cells/well and

grown for three days before testing. Cells were washed twice in D-PBS, then overlaid with test

compounds in HBSS at indicated concentrations in duplicate, as well as positive control inhibitor

and blank HBSS wells, and incubated at 37ºC for 30 min. Buffer was aspirated off cells and

dt

dM

CAP r

Dapp *

)0(*

1 =


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replaced with identical drug concentrations containing 0.1 µM calcein AM. Fluorescence was

immediately scanned (t = 0 min) using XFluor4 v4.40 software on a Safire fluorescent plate

reader (TECAN, Durham, NC) set at excitation wavelength 488 nm, emission wavelength 535

nm. The plate was incubated at 37ºC for 40 min, followed by a second scan (t = 40 min).

Percent inhibition was calculated from the difference between the two scans, using positive

control data as maximum inhibition (100%), and an IC50 was generated for each test compound

using nonlinear regression with GraphPad Prism software.


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Results

Pharmacokinetics. The pharmacokinetics of all treatment groups are summarized in Table 1.

During the in-life phase of the study no clinical signs of changes in health or behavior were

observed in any subject or treatment group. Ivermectin (T01) was readily absorbed in beagles

from the glycerol formal/propylene glycol formulation, producing a mean Tmax,obs value similar

to chewable formulations (Daurio et al., 1992) (Fig. 1). Cmax,obs, Tmax,obs, CL/F, and AUC0-∞ were

in excellent agreement with previous cross-bred dog studies utilizing the same liquid formulation

after correction of differences in dose (Gokbulut et al., 2006). The mean terminal elimination

half-life of ivermectin (106 h) was determined to be somewhat longer than previously reported,

which ranged from ~37-80 h (Lo et al., 1985; Kojima et al., 1987; Gokbulut et al., 2006). Time

after dosing that ivermectin remained detectable in plasma appeared to have some influence over

the calculated terminal t1/2 in these previous studies due to the multi-compartmental kinetic

profile.

Plasma spinosad concentrations, measured as the primary spinosyn A constituent, indicated rapid

absorption from its chewable formulations (Table 1). Like ivermectin, spinosad demonstrated a

long terminal half-life (Fig. 1).

Co-administration of ivermectin and spinosad resulted in significant changes in plasma

concentrations and pharmacokinetic parameters for ivermectin, but not for spinosad. Plasma

ivermectin concentrations in T03 were significantly higher than the control T01 from 6 h to the

last time point above the limit of quantitation. Cmax,obs was also higher (p=0.0298) than the

control group Cmax,obs value by 2.5-fold on average. Furthermore, the mean AUC0-∞ for


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ivermectin was 3.6-fold higher than the control group on average during dosing with spinosad

(p=0.0010). The mean CL/F for ivermectin was decreased 3.8-fold in the presence of spinosad

(p=0.0013). For spinosad, no statistical difference was observed in mean AUC0-∞, Cmax,obs, t1/2, or

Tmax,obs, (p>0.1) when administered with ivermectin (T03) as compared to the absence of

ivermectin (T02). One subject in T03 demonstrated delayed absorption of spinosad, but not

ivermectin (data not shown). This prevented the characterization of standard deviations in

plasma spinosad concentrations before the 6 h time point.

P-gp-mediated efflux. P-gp mediated efflux of spinosad was measured in MDCK-MDR1 cells

over a range of concentrations, along with the well characterized P-gp substrate digoxin at 2 µM.

Both compounds were also tested in MDCKLE cells, a subpopulation of MDCK II wild-type cells

with low P-gp efflux function. As shown in Table 2, transport was not polarized in MDCKLE

cells for either compound, with efflux ratios at unity. Digoxin showed strongly polarized

transport in MDCK-MDR1 cells; this was completely eliminated by ivermectin or by spinosad,

at 10 µM each. Polarized efflux was seen for spinosad at very low concentrations (less than 2

µM) in MDCK-MDR1 cells, with an efflux ratio of approximately 3.5. However as

concentrations increased apparent saturation of the efflux pump occurred, preventing P-gp

function and resulting in decreasing efflux ratios. Spinosad efflux at 0.5 µM was also blocked

by ivermectin, and cyclosporin A, a known P-gp inhibitor.

The potent inhibition of P-gp-mediated digoxin efflux by spinosad and the saturation of spinosad

transport by P-gp at low concentrations suggested that spinosad was a high affinity P-gp ligand

that could mediate P-gp drug interactions. Spinosad inhibition of P-gp was further examined in


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the calcein AM accumulation assay, in parallel with ivermectin. Ivermectin has been previously

shown to inhibit P-gp (Didier and Loor, 1996). Calcein AM is freely taken up by passive

permeability across cell membranes but is rapidly effluxed from the membrane as a substrate of

P-gp. Once internalized, however, calcein AM is irreversibly cleaved by nonspecific

endogenous esterases to produce the fluorescent free acid calcein. Inhibition of the P-gp

transporter results in the accumulation of calcein within the cell and a corresponding increase in

cellular fluorescence (Tiberghien and Loor, 1996). A proprietary Pfizer compound, CP100356,

has been validated as a potent P-gp inhibitor with maximal inhibition at 5 µM (Kalgutkar et al.,

2009). The inhibition of P-gp with 5 µM CP100356 was used as a 100% inhibitory control,

which was then used to compare calcein AM inhibition by ivermectin and spinosad. Figure 2a

shows that ivermectin and spinosad are similar in their human P-gp calcein AM inhibition

potency, with IC50 values of 1.19 and 3.23 µM (1041 and 2364 ng/mL), respectively. In

addition, spinosad was shown to inhibit the human P-gp basolateral to apical transport of

ivermectin (1 µM) with an IC50 of 2.3 µM or 2013 ng/mL (Figure 2b).


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Discussion

The transmembrane ATP-binding cassette transporter P-gp is known to influence drug

absorption in the intestinal tract, distribution to the central nervous system, tumors, and other

tissues, and to influence drug elimination by the liver and intestinal tract (reviewed by (Ho and

Kim, 2005)). As a result, P-gp may influence the efficacy and safety of a drug, or cause an

interaction with other co-administered drugs.

P-gp-sensitive drug pharmacokinetics and drug interactions have been studied in dogs for only a

few drugs, though inferences have been drawn from other species (Martinez et al., 2008).

Probably the most well-known examples of P-gp-sensitive pharmacokinetics in dogs have been

associated with a coding sequence 4-base pair deletion beginning at nucleotide 294 of the dog P-

gp gene (Mealey et al., 2001). In dogs and other mammals, ivermectin toxicity is caused in part

by activation of GABA receptors, but at low doses P-gp prevents CNS accumulation of

ivermectin. Characterization of brain tissue ivermectin concentrations in ivermectin-sensitive

collies (Pulliam et al., 1985) supports a link between P-gp function and the CNS distribution of

ivermectin. The CNS disposition of ivermectin and related macrocyclic lactones has also been

linked to P-gp function in rodents (Schinkel et al., 1994; Kwei et al., 1999; Kiki-Mvouaka et al.,

2010).

The impetus for the current study was an FDA warning on co-administration of spinosad with

extra-label high doses of ivermectin, which resulted in ivermectin-like toxicities in dogs.

Interestingly, the FDA findings did not mention any breed effects (i.e. collies) that might

implicate variants of P-gp. However, if spinosad were to be confirmed as a P-gp inhibitor, then


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the increased incidence of ivermectin toxicity observed with concurrent spinosad treatment could

be explained by a P-gp drug-drug interaction. For instance, spinosad inhibition of ivermectin

efflux at the BBB could result in increased CNS penetration of ivermectin and GABA-associated

adverse events in P-gp wild-type dogs.

After conducting several in vitro cellular assays commonly used to assess P-gp transport and

inhibition, spinosad was confirmed to be a substrate and inhibitor of human P-gp using

established substrates and ivermectin. However, the question of species differences in P-gp

ligand recognition and kinetics must be considered even though the human P-gp amino acid

sequence is calculated to be 90.3% identical to dog P-gp. Yokoi and colleagues have

demonstrated several cases where P-gp ligands have different affinities between species, though

human P-gp substrates and inhibitors were also dog P-gp substrates and inhibitors (Katoh et al.,

2006; Suzuyama et al., 2007). Further complicating the question of species differences are the

current limitations to characterizing dog-specific P-gp activity and inhibition. Cell-based

systems for dog P-gp are not commercially available, though some contract research companies

offer dog P-gp screening service. Beagle P-gp membranes are commercially available, but are

only useful for showing inhibition of ATPase activity, and are not well-suited to establishing

transport or interactions with substrates that do not produce high ATPase activity.

While suggestive of an ivermectin-spinosad interaction, the use of human P-gp in in vitro

screening was not considered definitive. Therefore, an in vivo study was next conducted to

further probe the potential for drug interaction. A 10X heartworm prevention dose of ivermectin

was administered as a liquid formulation rather than a chewable formulation to achieve a dose


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that would enable detection of plasma drug concentrations. The liquid formulation is further

postulated to mimic demodicosis treatment in the clinic where the high doses of ivermectin

needed are not easily achieved with chewable formulations. Statistical analysis demonstrated

significant increases in the AUC and Cmax,obs values for ivermectin upon dosing with spinosad

(Figure 1a; Table 1). A lack of ivermectin toxicity in T03 is attributed to the low, single dose of

ivermectin used (~60 µg/kg). Even with a spinosad interaction, it can be estimated that the

ivermectin plasma exposures in our study would not reach levels achieved during repeated

demodicosis treatments (~300 µg/kg/d).

Several laboratories have used various mice strains or knockouts to demonstrate at least a partial

role of P-gp inhibition in intestinal drug interactions involving ivermectin (reviewed in (Chen et

al., 2003; Murakami and Takano, 2008; del Amo et al., 2009)). Ivermectin is eliminated mostly

unchanged in bile/feces (Gonzalez Canga et al., 2009), and so as a P-gp substrate, it is a

candidate for secretion by the liver or intestine. Accordingly, the possibility that systemic

ivermectin concentrations increase due to spinosad inhibition of secretion should be considered,

especially as ivermectin is known to undergo P-gp-mediated exsorption in rodents (Sparreboom

et al., 1997; Laffont et al., 2002; Ballent et al., 2006). Alternatively, the increase in the dog

ivermectin Cmax and lack of change in the terminal half-life could suggest spinosad increased the

oral fraction of ivermectin absorbed. This could occur either by inhibiting ivermectin

metabolism or inhibiting its intestinal efflux. Unfortunately, oral ivermectin bioavailability data

is scarce and a primary reference was not available for dog bioavailability. Also, published

intravenous data does not include AUC values from which to estimate Foral. One reference does

cite the oral bioavailability of ivermectin in dogs as being high (95%) (Plumb, 2005), so if


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accurate, the inhibition of intestinal efflux would not greatly increase the fraction absorbed and

the first-pass metabolism must be minimal. In fact it was previously shown that only low levels

of the 3-O-desmethyl metabolite have been found in dogs and that the potent P-gp inhibitor

ketoconazole did not alter metabolism of ivermectin in dogs despite increasing its AUC and Cmax

(Hugnet et al., 2007). These findings lend support to the secretion inhibition model. However,

in this scenario, pharmacokinetic theory would predict Cmax, Tmax, and half-life to increase if

inhibition of systemic elimination were responsible for the drug interaction. Spinosad did not

increase the oral ivermectin Tmax or half-life (Table 1), but the pharmacokinetics do not

necessarily rule out inhibition of ivermectin secretion. For instance, inhibition of hepatic

canalicular P-gp secretion may have relatively modest effects on the plasma Tmax or half-life for

drugs excreted in bile (i.e. ivermectin) since inhibiting biliary elimination is predicted to impact

liver concentrations to a greater degree than systemic drug concentrations (Watanabe et al.,

2009). However, until more intravenous data is available, the possibility that spinosad increases

the fraction of oral ivermectin absorbed by inhibiting intestinal efflux cannot be discounted.

Two final discussion points deserve comment. First, ivermectin did not alter spinosad

pharmacokinetics even though it might be expected since spinosad is a P-gp substrate and, like

ivermectin, does not appear to be extensively metabolized in rats or livestock (Fed.Regist., 1999;

Rutherford et al., 2000). It is proposed that the large difference in spinosad and ivermectin doses

and corresponding drug exposures likely prevented the observation of a spinosad interaction.

Therefore, plasma protein binding of the two drugs could also be relevant. Second, the ability of

the spinosad formulation to enhance ivermectin absorption cannot yet be ruled out. A

formulation mimicking the one used in the commercial product could not be reproduced to serve


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as a control for these studies. However, formulation effects are considered to be less likely than

a drug-drug interaction based on the P-gp activity and inhibition profiles of spinosad.

In summary, the results of these studies demonstrate that spinosad causes a pharmacokinetic

interaction with ivermectin in dogs and that the interaction is likely due to inhibition of P-gp as

assessed in vitro using human P-gp. Based on these findings, it is the P-gp inhibition by

spinosad that is hypothesized to increase the risk of ivermectin toxicoses during high dose

ivermectin treatments, though the extent of BBB P-gp involvement remains in question. On one

extreme, increased systemic ivermectin exposures may simply translate to higher CNS drug

exposures even if inhibitory concentrations of spinosad are not present in the BBB. Conversely,

it may be that it is the local BBB inhibition of P-gp that drives CNS penetration of ivermectin.

Further complicating the issue is the possibility that multi-drug resistance-associated proteins,

organic anion-transporting polypeptides, and other transporters are co-localized with P-gp that

may also have ivermectin and spinosad activities that contribute to the drug interaction. Hence,

further studies, including CNS drug distribution, would be prudent to determine the dose

adjustment(s) that will ensure the safe administration of combination high dose ivermectin-

spinosad therapies without altering efficacy of either medication.


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Acknowledgements

The authors wish to thank T. Cox, M. Aaron, T. Flook, E. Vera, S. Strombeck, R. Fowler, and T.

Vandegiessen of the Pfizer Animal Health Animal Research Support Group for their technical

expertise. We also thank Drs. S. Brown, D. Merritt, and J. Robinson for support of this project,

Dr. D. Billen for helping obtain spinosad analogs, Drs. S. Cox and W. Collard for thoughtful

review of the manuscript, and Dr. M. Troutman for providing background information on cell

lines.


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Authorship contribution

Participated in research design: Dunn, Hedges, Lai, Mahabir, Balogh, Locuson

Conducted experiments: Dunn, Sampson, Balogh

Contributed analytic tools: Hedges

Performed data analysis: Hedges, Sampson, Lai, Mahabir, Balogh, Locuson

Wrote or contributed to the writing of the manuscript: Dunn, Hedges, Sampson, Lai, Mahabir,

Balogh, Locuson


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Footnotes

b) This work was conducted in partial fulfillment of a Professional Science Masters Degree in

Integrative Pharmacology at Michigan State University (author STD).

c) Reprints:

Charles W Locuson

Pfizer Animal Health, Veterinary Medicine Research and Development, Metabolism & Safety,


T: 269-833-2505

F: 269-833-7721

[email protected]

Current author affiliations:

1Life Technologies, Austin, TX

2Pfizer, Groton, CT


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Figure Legends.

Figure 1. Plasma pharmacokinetic profiles of ivermectin and spinosad dosed separately (�) or

with each other (�). Total mean plasma concentration values in ng/mL are shown with standard

deviations. The first 24 h after dosing are shown inset.

Figure 2. (a) Calcein AM assay for P-gp inhibition. MDCK-MDR1 cells are treated with

CP100356 (�), spinosad (�), or ivermectin (▼) in HBSS at indicated concentrations for 30 min

prior to adding calcein AM for an additional 40 min. Fluorescence was measured using

wavelengths for excitation at 488 nm, emission at 535 nm. Increased intracellular fluorescence is

directly proportional to P-gp inhibition. Taking the fluorescence value for 5 µM CP100356 as

equal to 100% inhibition, the percent inhibition was calculated for spinosad and ivermectin.

Dashed line indicates 50% inhibition, or IC50. (b) Spinosad inhibition of ivermectin efflux by P-

gp (Mean ± standard deviation). Data were fit as described in the Methods.


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Table 1. Plasma pharmacokinetic summary of ivermectin, spinosad, and concomitant ivermectin/spinosad treatments in healthy beagle dogs. T01 T02 T03 Drug Ivermectin Spinosad Ivermectin Spinosad Dose (mg/kg) a,b0.0815 ± 0.00319 38.0 ± 7.0 b0.0562 ± 0.0211 40.8 ± 5.2 Cmax,obs (ng/mL) 24.0 ± 6.6 1550 ± 356 c60.1 ± 20.9

(p=0.0298) 2100 ± 904

Tmax,obs (h) 5.3 ± 4.0 3.3 ± 2.3 5.0 ± 4.4 5.0 ± 4.4 t1/2 (h) 106 ± 33 271 ± 30 140 ± 11 265 ± 51 AUC0-∞ (ng×h/mL) 934 ± 241 55900 ± 23900 c3400 ± 538

(p=0.0010) 62500 ± 15700

CL/F (mL/h/kg) 67.3 ± 15.1 571 ± 183 c17.7 ± 2.5 (p=0.0013)

523 ± 141

aAll values represent the mean ± standard deviation. bIvermectin doses were calculated using the density of the most abundant formulation excipient, propylene glycol (d=1.036 g/mL). cDenotes a statistical difference (p<0.10) was observed during co-administration of drugs relative to single drug administration as described in the Methods.


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Table 2. Efflux of digoxin and spinosad in MDCKLE or MDCK-MDR1 cells in bidirectional assay. Compounds were tested at indicated concentrations in each direction in triplicate. Transport proceeded for 2 hours, followed by measurement of analyte in donor and receiver chambers. Compound Concentration (µM )

Inhibitor Cell line Efflux Ratio

(BA/AB)

Digoxin

2 MDCKLE 0.91 2 MDCK-MDR1 13.86 2 aIvermectin MDCK-MDR1 1.64 2 aSpinosad MDCK-MDR1 1.34

Spinosad (Spinosyn A)

2 MDCKLE 0.85 0.1 MDCK-MDR1 3.58 0.5 MDCK-MDR1 3.45 2 MDCK-MDR1 1.11

10 MDCK-MDR1 0.50 100 MDCK-MDR1 0.34 0.5 bCyclosporin A MDCK-MDR1 0.70 0.5 aIvermectin MDCK-MDR1 0.82

aIncluded in apical and basolateral chambers at 10 µM. bIncluded in apical and basolateral chambers at 20 µM.


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