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Small Ruminant Research 93 (2010) 103–109

Contents lists available at ScienceDirect

Small Ruminant Research

journa l homepage: www.e lsev ier .com/ locate /smal l rumres

nfluence of verapamil on pharmacokinetics and transplacentalransfer of ivermectin in sheep

ubén Péreza,∗, Cristina Palmaa, María-José Núneza, Pedro Urrutiaa, Aurelio Salazara,uis Moralesa, Daniela Veraa, José Coxb

Departamento de Ciencias Clínicas, Facultad de Ciencias Veterinarias, Universidad de Concepción, Chillán, ChileDepartamento de Ciencias Pecuarias, Facultad de Ciencias Veterinarias, Universidad de Concepción, Chillán, Chile

r t i c l e i n f o

rticle history:eceived 26 August 2009eceived in revised form 6 April 2010ccepted 19 May 2010vailable online 12 June 2010

eywords:-glycoproteinerapamil

vermectinharmacokineticsetal sheep

a b s t r a c t

In pregnant sheep at 120–128 days of gestational age, a study was done to evaluate theeffect of the co-administration of verapamil (Vpm) and ivermectin (IVM) on the mater-nal and fetal disposition kinetics after intravenous administration of IVM. Ten pregnantSuffolk Down sheep of 63.5 ± 6.6 kg body weight (bw) were surgically prepared to insertpolyvinyl catheters in the fetal femoral artery and vein and amniotic sac. The ewes wererandomly assigned to two experimental groups. In Group 1 (control), 5 ewes were treatedwith an intravenous bolus of 0.2 mg IVM/kg bw. In Group 2 (Vpm-IVM), 5 ewes were sub-cutaneously treated with Vpm (2.5 mg/kg × 3 doses at 12 h intervals) and 0.2 mg/kg IVMby intravenous route. Maternal and fetal blood samples were taken before and after IVMadministration during a 144 h post-treatment period. Samples were analyzed by liquidchromatography (HPLC). A non-compartmental pharmacokinetic analysis was performedand statistical differences were determined using the Mann–Whitney U-test.

Significantly higher IVM levels in maternal plasma concentrations were determined afterco-administration of Vpm/IVM compared with the group treated with IVM alone. Signifi-

cant decreases in the volume of distribution and in the half-life of elimination (t½ˇ) wereobserved in the Vpm/IVM treated group. A significantly faster (Tmax) and higher (Cmax)(P < 0.05) increase in IVM fetal plasma concentrations were observed in the group of fetusesfrom pregnant ewes treated with Vpm/IVM. The results of our study support the hypothesisthat pharmacological blockage of P-glycoprotein with Vpm can increase the transfer of IVMto fetal circulation.

. Introduction

IVM is a member of the family of compounds pro-uced by the soil microorganism Streptomyces avermitilisnd known generally as avermectins. The name indicateshe vermicidal and ectoparasiticidal properties of the group

∗ Corresponding author at: Laboratorio de Farmacología, Facultad deiencias Veterinarias, Universidad de Concepción, PO Box 537, Chillán,hile. Tel.: +56 42 208826; fax: +56 42 275302.

E-mail address: rubperez@udec.cl (R. Pérez).

921-4488/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.smallrumres.2010.05.010

© 2010 Elsevier B.V. All rights reserved.

(Campbell and Benz, 1984). The large scale use of IVM inman and target animals over the years has provided evi-dence for the safety of the drug at the applied dose rates(Burkart, 2000). At very high doses, however, neurointoxi-cation may develop with symptoms such as ataxia, tremors,depression and death (Lankas and Gordon, 1989; Lankas etal., 1997). This neurotoxicity is presumed to involve IVM

binding to GABA-gated chloride channels, which are con-fined to the central nervous system in mammals (Turnerand Schaeffer, 1989). It has been shown that at normaldoses IVM reach very low concentrations in the brain (Chiuand Lu, 1989). The reason for this low distribution has

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104 R. Pérez et al. / Small Rumi

been attributed to the presence of a drug transporter atthe blood–brain barrier called P-glycoprotein which pumpsIVM back into blood (Schinkel et al., 1994; Didier and Loor,1995).

P-glycoprotein (P-gp) is a 170-kDa membrane proteinthat functions as an adenosine triphosphate-dependentdrug-efflux pump, actively secreting a variety of drugs andtoxins from cells (Fromm, 2004; Leslie et al., 2005). Expres-sion of P-gp has been identified in the capillary endothelialcells of the brain (blood–brain barrier), intestine, kidney,liver, testes and placenta (Ceckova-Novotna et al., 2006;Hennessy and Spiers, 2007). These tissues are extremelyimportant for the absorption, distribution and eliminationof drugs. Moreover, recent studies indicate that many com-monly prescribed drugs are substrates of P-gp, and many ofthem also inhibit its function (Sikic et al., 1997; Baylassacet al., 2005; Wang et al., 2003). The ability to extrude xeno-biotics gives this protein some physiological properties ofprotection and detoxification. In the mouse, placental P-gphas been shown to have an important role in protecting thefetus from a number of different molecules including syn-thetic steroids and agricultural/domestic herbicides andpesticides (Sun et al., 2006). Further, placental P-gp defi-ciency resulted in 100% fetal susceptibility to cleft palateinduced by low doses of IVM in mice (Schinkel et al., 1994).Smit et al. (1999) showed that transplacental transfer ofthe P-gp substrates digoxin, saquinavir and paclitaxel was2.4–16 times higher in P-gp (mdr 1a/1b) knockout micecompared to mice with normal P-gp function. Moreover,the P-gp inhibitor PSC833 and GG918 caused a 5- to 7-fold increase in the placental transfer of saquinavir in micewith normal P-gp function but not in mdr1a/1b knockoutmice, lacking functional P-gp. These results support thehypothesis that P-gp may play an important role limitingthe fetal penetration of potentially harmful or therapeuticcompounds. Hence, this P-gp function could be removedby pharmacological means (Smit et al., 1999).

Since the discovery of Vpm’s ability to reverse P-gpmediated drug resistance, much research work has beendevoted to the investigation of drug binding sites and themechanisms by which various modulators may inhibit P-gp function. It was found that Vpm can both stimulate (ifthe pump is inactive) and inhibit (if actively pumping) theactivity of P-gp. In addition, Vpm can binds reversibly toP-gp, hence inhibiting the binding of many chemothera-peutic agents as well as other modulators to P-gp (Wanget al., 2003). Thus, it is evident that drug–drug interactionswith P-gp are possible. These interactions would lead to anincrease in drug levels, firstly in the blood compartment,and secondly, in organs protected by blood–tissue barri-ers, which may lead to an increase in the toxicity of thespecific drug (Baylassac et al., 2005). Our working hypoth-esis is that the pharmacological blockage of P-gp with Vpmcan increase the transfer of IVM to the fetal circulation. Toaddress this assumption, we conducted a study in preg-nant sheep between 120 and 130 days of gestational age

to characterize the pharmacokinetics and transplacentalexchange of IVM. The aim of the study was to evaluatethe effect of the co-administration of Vpm and IVM onthe maternal and fetal disposition kinetics of IVM after theintravenous administration in pregnant sheep.

earch 93 (2010) 103–109

2. Materials and methods

2.1. Animals and management

A flock of 10 pregnant Suffolk Down ewes, clinically healthy, between120 and 130 days of gestational age and mean body weight (BW) of63.5 ± 6.6 kg was used for the study. The flock was housed in collectivepens on a dry bed, fulfilling individual requirements of space, ventila-tion, and free access to fresh water. Ewes were fed daily with alfalfa hayand supplemented with grain (400 g of barley per ewe) and water wasprovided ad libitum.

2.1.1. Drugs and reagentsPure standard IVM (mainly 22,23 dihydroavermectin B1a) and Vpm

were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All reagentsused for the extraction and chromatographic analysis of plasma sampleswere of HPLC grade (Merck, Darmstadt, Germany).

An experimental formulation of 0.5% IVM for maternal intravenousadministration, was prepared diluting a fixed volume of the commercialformulation at 1% of IVM (Ivomec® , Merial, Haarlem, The Netherlands) ina solution that contained a mixture of propylene glycol, ethanol and ster-ile distilled water (13, 27 and 60%, v/v, respectively). Vpm was diluted inthe same solution as IVM. The following formulations were used duringsurgical procedures: atropine (0.02 mg/kg; Atropina sulfato, LaboratorioChile, Santiago, Chile); acepromazine (0.02 mg/kg; Pacifor® , Drag PharmaInvetec, Santiago, Chile); ketamine (5 mg/kg; Ketostop® , Drag PharmaInvetec); halothane (Halotano, Cristália Productos Químicos Farmacéu-ticos Ltda, Itapira, Brasil), heparin (Heparina sódica, Laboratorio Biosano,Santiago, Chile); sodium benzypenicillin (Penicilina G Sódica, LaboratorioVeterquímica, Santiago, Chile); gentamicin (Gentamicina al 10%, Labora-torio Chile); flunixin meglumine (Febrectal® , Drag Pharma Invetec).

2.2. Surgical procedures

Eight Suffolk Down pregnant ewes between 120 and 128 days ofgestation were surgically prepared for the study using to the procedurepreviously reported by Perez et al. (1989), with modifications in the anes-thetic protocol. Briefly, following food and water deprivation for 24 h,ewes were pre-medicated with 0.02 mg/kg of atropine and 0.02 mg/kg ofacepromazine by the intramuscular route. Thirty minutes after tranquil-ization, ewes were anesthetized with 5 mg/kg of ketamine and then wereintubated using an endotracheal tube. Anesthesia was maintained usinghalothane (vaporizer setting 2–3%) and 100% oxygen. Once anesthetized,the ewes were prepared for surgery and then polyvinyl catheters contain-ing heparinized saline solution were inserted into fetal femoral artery andvein and into the amniotic sac (Pérez et al., 2008).

A serum clinical biochemistry panel, including hepatic test, was per-formed to evaluate the animals’ health and once the values were withinthe normal ranges described for the ovine species, they were subjected tothe experimental treatments.

3. Animal treatment

The animals were purposely assigned into two exper-imental groups. In Group 1 (IVM alone), 5 ewes weretreated with an intravenous dose of 0.2 mg IVM/kg bodyweight, administered through the maternal jugular vein.Considering both the risk for fetal survival to the surgi-cal preparation and the ethical recommendations to avoidunnecessary suffering of animals, the data for this groupof pregnant sheep were taken from a previously publishedreport by our laboratory (Pérez et al., 2008). This reportdescribes the maternal and fetal pharmacokinetics of IVMafter its intravenous administration in pregnant sheep. In

Group 2 (Vpm-IVM), 5 ewes were pre-medicated with 3subcutaneous doses of 2.5 mg/kg of Vpm at 12 h inter-vals; i.e. at 8:00 PM the previous day, 8.00 AM and 8:00 PMof the treatment day. The effective dose of 3 mg/kg ofVpm was taken as reference using considerations reported

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y Molento et al. (2004). Furthermore, considering thever-estimation that produce the gravid uterus on bodyeight of pregnant sheep, a dose of 2.5 mg/kg was con-

idered as the most appropriated for this study. Ivermectin0.2 mg/kg) was injected by the intravenous route at 30 minfter the second dose of Vpm. Care was taken to maintainhe ewes of Group 2 under same management and experi-

ental conditions as observed in ewes of Group 1.

.1. Sampling procedures

In both groups, maternal and fetal blood samples wereaken before IVM administration from the dam (5 mL) androm the fetus (1.5 mL) as a control. After IVM administra-ion, maternal and fetal samples were taken at 0.25, 0.5,.0, 2.0, 4.0, 8.0, 12.0, 24.0, 48.0, 72.0, 96.0, 120 and 144 host-administration. Blood samples were taken by directuncture of the jugular vein of the dam and through theatheter inserted in the femoral artery of the fetus. Samplesere collected in heparinized tubes and then centrifuged

or 15 min at 2000 × g to obtain plasma. The plasma wastored in polypropylene tubes and frozen at −18 ◦C.

All procedures were performed with the authorizationf the Ethical Committee for Animal Experimentation of theacultad de Ciencias Veterinarias, Universidad de Concep-ión, Chile.

.2. Analytical procedures

Plasma concentrations of IVM were assayed by high per-ormance liquid chromatography (HPLC) with fluorescenceetection after solid phase extraction (SPE), according torocedures described elsewhere (Perez et al., 2008).

.3. Drug extraction and derivatization

Drug-free 1-mL maternal plasma samples were forti-ed with IVM to reach final concentrations of 0.1, 0.5,.0, 5.0, 10.0, 25.0, and 50.0 ng/mL. Fetal plasma samplesere fortified using drug concentrations in the range of

.1–10 ng/mL. Fortified and experimental plasma samplesere vortexed for 30 s and then incubated for 15 min at

oom temperature, before SPE procedure was performedsing methods described by Pérez et al. (2008). Briefly, 1 mLf acetonitrile and 0.25 mL of water were added to 1 mL oflasma. After SPE, the eluate was evaporated to drynessnder a gentle stream of nitrogen at 60 ◦C in a water bath.he residue was subjected to derivatization according tohe procedures described by De Montigny et al. (1990);or this purpose the residue was dissolved in 100 �L of-methylimidazole solution in acetonitrile (1:1, v/v). To

nitiate the derivatization, 150 �L of trifluoroacetic anhy-ride solution in acetonitrile (1:2, v/v) was added. Afterompletion of the reaction (<30 s), an aliquot (100 �L) ofhis solution was injected directly into the chromatograph.

.4. Chromatographic conditions

Maternal and fetal IVM plasma concentrations wereetermined with the use of a Shimadzu 10AVP HPLC system

earch 93 (2010) 103–109 105

(Shimadzu, Kyoto, Japan). The HPLC analysis was under-taken with the use of a Supelcosil C18 column (3 �m;4.6 mm id × 150 mm; Supelco INC) kept in a column ovenat 30 ◦C (CTO-10AS VP; Shimadzu) and a fluorescencedetector (Spectrofluorometric detector RF-10; Shimadzu)reading was taken at an excitation wavelength of 383 nmand an emission wavelength of 447 nm. The mobile phaseconsisted of acetic acid (0.2% in water), methanol, andacetonitrile (4:32:64, v/v/v) passing at a flow rate of1.5 mL/min.

3.5. Method of calibration

Calibration curves for IVM in the range 0.1–50 ng/mLwere prepared with the use of drug-free maternal plasmaof non-treated ewes, whereas for fetal plasma the cali-bration curve was prepared in the range of 0.1–10 ng/mL.Pooled plasma samples were taken throughout the proce-dure, and calibration curves were plotted with the use ofthe peak area as a function of analyte concentration. Linearregression analysis was used to determine the slopes andcorrelation coefficients of the different calibration curves(n = 5). The extraction efficiency of the drug under studywas measured by comparison of the peak area of the spikedplasma samples, against the peak area resulting from directinjections of the IVM standard in methanol. The interassayprecision of the PSE and HPLC procedures was evaluatedby processing in quintuplicate aliquots of plasma samplescontaining known amounts of the drug on different days.

The analytical method used to extract, derivatize, andquantify plasma concentrations of IVM by HPLC analysiswas validated as follows: the regression lines between peakareas and drug concentrations showed correlation coef-ficients (r2) ranging from 0.9993 to 0.9998 for maternaland fetal plasma, respectively. The mean extraction recov-ery from maternal plasma was 82.8 ± 6.4% at the spikedconcentrations between 0.1 and 50 ng/mL. The recoveryvalue for the fetal plasma was 92.3 ± 3.7%. The interas-say precision showed variation coefficients of 3.6 ± 1.4 and1.6 ± 0.5% for maternal and fetal plasma, respectively. Thelimit of drug detection (LOD) was established by injectionof maternal or fetal plasma blanks and the measurementof the baseline noise at the retention time of IVM peak. Themean baseline noise of IVM at the retention time plus threestandard deviations was defined as the theoretical detec-tion limit. The LOD for both maternal and fetal plasma wasfound to be 0.05 ng/mL.

3.6. Data analysis

Pharmacokinetic parameters were determined bymeans of a non-compartmental model using computersoftware (PK Solutions, Summit Research Services, Ash-land, OH, USA). The peak concentrations (Cmax) andtime-to-peak concentrations (Tmax) were read from theplotted concentration–time curve in each individual

animal. The terminal (elimination) half-life (t½ˇ) was cal-culated as ln 2/ˇ, where ˇ represents the first order rateconstant of IVM elimination. The ˇ value was deter-mined by performing regression analysis using at least fivepoints of the terminal phase of the concentration–time

106 R. Pérez et al. / Small Ruminant Research 93 (2010) 103–109

treatment groups, although slight differences in the slopeof both curves are observed (Fig. 1).

The co-administration of Vpm with IVM caused signif-icant changes in the pharmacokinetics variables of IVM

Fig. 1. Maternal plasma concentrations (mean ± SD) of IVM after itsmaternal intravenous administration either alone or co-administeredwith Vpm to pregnant sheep.

plot. The area under the plasma concentration–time curve(AUC) was calculated by the trapezoidal rule and furtherextrapolated to infinity by dividing the last experimentalconcentration by the terminal slope. Statistical momenttheory was applied to calculate the mean residence time(MRT) using the formula:

MRT = AUMCAUC

where AUMC is the area under the curve of the productof time and drug concentration versus time from zero toinfinity, and AUC is the area under the plasma concen-tration–time curves, as previously defined. The volume ofdistribution (Vd(area)) was estimated using the formula:

Vd = DoseAUC · ˇ

And the total body clearance (ClB) was calculated by:

ClB = DoseAUC

Pharmacokinetic parameters were reported asmean ± SD and were compared using either ANOVAor the nonparametric Mann–Whitney test. All statisticalanalyses were performed using Graph Pad Instat softwarev. 3.0 (Graph Pad Software Inc., San Diego, CA, USA). Meanvalues were considered significantly different at P < 0.05.

4. Results

No adverse effects of the treatments were observedin the two groups of animals throughout the experi-mental period. Maternal and fetal plasma concentrationsafter maternal intravenous injection of IVM alone or co-administered with Vpm in pregnant sheep, are shown inFigs. 1–3. The maternal and fetal pharmacokinetic variablesare listed in Table 1.

4.1. Maternal plasma concentrations andpharmacokinetics of IVM

The IVM plasma concentrations measured up to 144 hafter the intravenous injection of IVM alone or co-

Fig. 2. Maternal plasma concentration of IVM over the first 8 h samplingtime after its intravenous administration either alone or co-administeredwith Vpm to pregnant sheep (*P < 0.05).

administered with Vpm in pregnant sheep are comparedin Fig. 1. IVM was detected in maternal plasma between15 min and 144 h after its intravenous administrationin pregnant sheep. Maternal plasma IVM concentrationsdecreased biphasically after intravenous administration(Fig. 1). In the group treated with IVM alone during thefirst phase the drug concentrations decreased exponen-tially from a mean value of 239.0 ± 33.0 ng/mL, at 15 minto 36.0 ± 8.7 ng/mL at 12 h post-administration. Whereas,in the group treated with IVM co-administered with Vpmplasma concentrations decreased from 393.1 ± 56.2 ng/mLat 15 min to 51.4 ± 21.4 ng/mL at 12 h post-treatment.Higher IVM plasma concentrations were determined dur-ing the first 4 h after co-administration of Vpm and IVMcompared with that of the IVM alone treatment (Fig. 2;P < 0.05). During the second phase the depletion curveof IVM plasma concentrations decreased linearly in both

Fig. 3. Fetal plasma concentrations of IVM (IVM) after its maternal intra-venous administration either alone or co-administered with Vpm topregnant sheep.

R. Pérez et al. / Small Ruminant Research 93 (2010) 103–109 107

Table 1Pharmacokinetic parameters for IVM in maternal and fetal circulation after maternal intravenous administration either alone or co-administered with Vpmto pregnant sheep (mean ± SD).

Pharmacokinetic parameter IVM Vpm–IVM P value

Maternal circulationAUC0–t (ng h/mL) 2925 ± 1076 3557 ± 680 0.525AUC0–∞ (ng h/mL) 3825 ± 1672 3985 ± 487 0.665t½ˇ

a (h) 70.9 40.6 0.033*

MRT (h) 99.2 ± 45.1 61.3 ± 33.5 0.030*

Vd (mL) 6054 ± 1402 3550 ± 670 0.007*

Clearance (ng mL/kg) 59.3 ± 25.9 57.9 ± 10.4 0.937

Fetal circulationCmax (ng/mL) 1.72 ± 0.6 4.14 ± 1.05 0.022*

Tmax (h) 8.0 ± 2.8 1.20 ± 0.45 0.007*

t½aba (h) 3.94 0.57 0.022*

AUC0–t (ng h/mL) 75.1 ± 11.4 96.0 ± 23.4 0.107AUC0–∞ (ng h/mL) 89.1 ± 10.9 117.8 ± 49.8 0.314t½ˇ

a (h) 46.8 50.9 0.905MRT (h) 77.6 ± 36.7 75.0 ± 37.0 0.913

Cmax, peak plasma concentration; Tmax, time-to-peak plasma concentration; AUC0–t , area under the concentration-time curve from t0 to the last measuredc o infinitr

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a Values r‘epresent the harmonic mean for t½ab and t½ˇ (days).* P < 0.05, for comparison between treatments.

Table 1). There were significant differences in the meanalues of plasma MRT and in the elimination half-life whenhese results were compared between treatments groups.he calculated values of Vd for the group treated withpm/IVM were lower (P < 0.05) than those obtained in theroup treated with IVM alone. The comparison of meanalues for AUC observed in both groups of sheep did nothow statistically significant differences. No significant dif-erences (P ≥ 0.05) in the mean values of clearance werebserved when both treatment groups were compared.

.2. Fetal plasma concentrations and pharmacokinetic ofVM

Ivermectin fetal plasma concentrations measured from5 min to 144 h after maternal administration in the fetusesf both treatment groups are shown in Fig. 3. A faster andarger (P < 0.05) increase in fetal plasma IVM concentra-ions was observed in the fetuses from Group 2 comparedhanges in plasma concentrations of fetuses from Group 1Fig. 3).

In agreement with the changes observed in plasma con-entrations, in the fetuses of Group 2, there were significantncreases (P < 0.05) in the IVM mean values of Cmax associ-ted to significant decreases (P < 0.05) in Tmax and in theetal half-life of absorption (Table 1). However, no signifi-ant changes were observed when the values of half-life oflimination, AUC and MRT, between both groups of fetusesere compared (Table 1).

. Discussion

Our study has shown that in the pregnant sheep the

dministration of Vpm caused marked changes in thelasma disposition kinetics of IVM. Together, our resultseem to indicate that a likely blockage of P-gp induced bypm would increase the placental transfer of IVM from theaternal to fetal circulation.

y; t½ab, half-life of absorption; t½ˇ , half-life of elimination; MRTC–∞ , mean

After the intravenous administration of IVM alone,plasma concentrations were in a range between239.0 ng/mL (15 min) and 36.0 ng/mL (at 144 h post-treatment). While in the group treated with the Vpm/IVMcombination, significantly higher plasma concentrationsof IVM were observed between 15 min and 4 h followingIVM administration. Similar results have been described inrats by Alvinerie et al. (1999) and in sheep by Molento et al.(2004). In both experiments the co-administration of Vpmand IVM induced an increase in plasma concentrationswhen the macrocyclic lactone was administered by topicalor oral routes. The intravenous route of administration wasused to avoid any interference of the absorption process onthe pharmacokinetic interaction between Vpm and IVM.

It has been demonstrated that IVM and related com-pounds interact with P-gp, which plays a pivotal role inthe bioavailability and tissue distribution of IVM in hostorganisms as well as in target parasites (Lespine et al.,2007). The P-gp located in the intestinal epithelium andbile canalicules contributes to the massive fecal elimina-tion of macrocyclic lactones (Laffont et al., 2002; Ballent etal., 2006; Hennessy et al., 2000). Thus, it has been proposedthat the enhanced plasma availability of IVM induced byVpm is due to its capacity to bind reversibly to intestinaland biliary canalicules P-gp and inhibit the binding of IVMto P-gp (Alvinerie et al., 2008; Wang et al., 2003).

The chief significant changes in the IVM pharmacoki-netic variables observed in the group of pregnant sheeptreated with Vpm/IVM were the increase in plasma con-centrations of IVM and the decrease in the volume ofdistribution. These findings were similar to those reportedby Lifschitz et al. (2002) for cattle treated with theloperamide/moxidectin combination. In addition, similar

results are described by Bansal et al. (2009), in rats inwhich Vpm was co-administered with the anticancer agentirinotecan. Alternatively, the short term increase in plasmaconcentrations of IVM in the pregnant sheep treated withthe combination Vpm/IVM, make possible to hypothesize

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108 R. Pérez et al. / Small Rumi

that the plasma disposition of IVM could also be influencedby the vasodilator effects of Vpm. It is well known thatVpm exerts its antihypertensive effects by both inducingperipheral vasodilatation and reducing peripheral vascu-lar resistance. These effects are mediated by the inhibitionof calcium influx into smooth muscle cells of the arterio-lar wall (Flekenstein, 1977). In addition, it is known thatin pregnant sheep during the last third of gestation thecardiovascular system undergoes important physiologicaladaptations to accommodate the growth and developmentof the fetus and the supporting placental unit (Frederiksen,2001). Such changes induce a state of high-flow and lowresistance of the cardiovascular system (Abbas et al., 2005).These changes may be increased by the vasodilator effectsof Vpm. These effects may induce a reduction in the bloodflow distribution to the tissues, causing an increase in thedisposition of IVM into the cardiovascular compartment ofthe pregnant sheep. Therefore, the increase in the rate ofelimination of IVM observed in sheep treated with Vpm,may be related to the cardiovascular modifications inducedby Vpm that decreased the volume of distribution of IVM.Accordingly, Ramirez et al. (1994) describe in rabbits thatthe intravenous administration of Vpm induces a diversionof blood flow towards the organs, preferentially towardsthe liver. Such changes can have a significant effect on theIVM elimination, inducing an increase in liver IVM concen-trations, thus facilitating its elimination through the biliarysecretion. In addition, due to IVM its high lipid solubility, itmay have a selective binding to the body fat tissue. Hence,fat may act as a reservoir reducing the rate of eliminationof the drug (Chiu and Lu, 1989).

From our results, it is not possible to know whetherthe enhancement of IVM plasma concentrations inducedby Vpm in pregnant sheep are due to an inhibitory effect onthe P-gp transport mechanism, or due to Vpm vasodilatoreffects modifying the hemodynamic of pregnant sheep. Acombination of both factors could be considered in furtherresearch.

In the group of sheep treated with IVM alone therewas a limited and slow transfer of IVM from the maternalblood towards fetal circulation. This transfer was charac-terized by lower fetal mean values of Cmax and higher Tmax

than the group of fetal sheep whose mothers were treatedwith the Vpm/IVM combination. Together these resultsmay indicate that a likely blockage of P-gp induced by Vpmwould produce a higher and faster placental transfer of IVMfrom the maternal to fetal circulation. Similar results weredescribed in rats by Ushigone et al. (2000) where the uptakeof vinblastine, vincristine and digoxin into the fetal circula-tion was significantly increased by P-gp inhibitors such ascyclosporine A and Vpm. These results are consistent withthe proposed role for the placental P-gp as a pharmaco-logical barrier against xenobiotics (Nekayeva et al., 2005;Nanovskaya et al., 2008). Furthermore, the protective roleof placental P-gp has been demonstrated in animal modelsin which the absence of P-gp in mouse placenta resulted in

a 100% susceptibility to IVM induced cleft palate (Lankas etal., 1998).

The transplacental transfer of IVM from the maternal tofetal circulation could pose a risk of attaining toxic concen-trations in the CNS of the fetus where the drug can produce

earch 93 (2010) 103–109

toxic effects. The CNS toxic effects of IVM are related to theIVM interaction with the mammalian GABAA receptor sys-tem. Nevertheless, in adult animals, due to the presenceof P-gp, macrocyclic lactones are excluded from the cere-brospinal fluid when the blood–brain barrier (BBB) is intact(Cotreau et al., 2003). However, this is not true for the fetusand the newborn in which the BBB has not yet fully devel-oped. In addition, because IVM shows high affinity for P-gp,the P-gp content of the placental barrier might pose a sig-nificant obstacle for the diffusion of IVM towards the fetus.Nonetheless, this barrier function accomplished by placen-tal P-gp, may be blocked by Vpm which could lead to IVMtoxic concentration in the fetal CNS. Due to risk involvedin the transplacental transfer of drug concentrations to thefetus, potentially posing a threat for fetal survival or normaldevelopment, a better understanding of P-gp drug–druginteractions in pregnant females are needed to be clarified.

In conclusion, the results of the study sustain the work-ing hypothesis that the pharmacological blockage of P-gpwith Vpm can lead to disruption of the blood–placental bar-rier and increase the transfer of IVM to the fetal circulation.

Acknowledgements

This study was supported by Research Grant FONDECYT1060986, CONICYT, Chile. The authors thank Dr. Marcos A.Munoz Domon for his valuable critical review of the article.

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