Influence of Verapamil on the Pharmacokinetics of The

Parasitol Res (2004) 92: 121–127 DOI 10.1007/s00436-003-1022-3

ORIGINAL PAPER

M. B. Molento Æ A. Lifschitz Æ J. Sallovitz Æ C. Lanusse R. Prichard Inﬂuence of verapamil on the pharmacokinetics of the antiparasitic drugs ivermectin and moxidectin in sheep

Received: 20 July 2003 / Accepted: 11 August 2003 / Published online: 21 November 2003 Springer-Verlag is a part of Springer Science+Business Media 2003

Abstract P-Glycoprotein (P-GP) is a transport protein resistant- or rate-limiting-parasites and on the persis- that participates in the mechanism of active secretion tency of its antiparasitic activity. of different molecules from the bloodstream to the gastrointestinal tract. The aim of the current work was to evaluate the effect of verapamil, a P-GP substrate, on the pharmacokinetic behaviour of the anthelmintics Introduction ivermectin and moxidectin in sheep. Thirty-two sheep were divided into four groups and treated orally with Ivermectin and moxidectin are 16-membered macrocy- either ivermectin or moxidectin alone (200 lg/kg) or clic lactones that are produced by fermentation of soil co-administered with verapamil at 3 mg/kg (three times moulds of Streptomyces spp. (Burg et al. 1979). Iver- at 12 h intervals). Blood samples were collected over mectin is a member of the avermectin family, having 30 days post-treatment and plasma was analysed to oleandrose residues, whereas moxidectin is a milbemycin determine ivermectin and moxidectin concentrations by without sugar residues. These avermectin and milbe- HPLC. The ivermectin peak concentration was signifi- mycin anthelmintics have a broad-spectrum activity cantly higher (P=0.048) after ivermectin plus verapa- against internal and external parasites in animals (Bogan mil, compared with the ivermectin alone treatment. and Armour 1987). Unfortunately, after just a few Ivermectin plasma availability was significantly higher generations of parasites being exposed to ivermectin, following co-administration (P=0.022). Verapamil had resistance was reported (Van Wyk and Malan 1988) and no effect on the kinetics of moxidectin. The significant has since become common in sheep and goats world- alteration in the plasma disposition of ivermectin in wide. In cattle, Cooperia spp. is often a dose-limiting sheep induced by verapamil, possibly due to interfer- nematode, which has recently been reported as having ence with a P-GP-mediated elimination mechanism, become resistant to ivermectin (Vermunt et al. 1995; may have an important impact on efficacy against Anziani et al. 2001; Coles et al. 2001; Fiel et al. 2001). Ivermectin and moxidectin are believed to act by binding to glutamate- and gammabutyric acid-gated chloride channels, resulting in a pharyngeal and somatic M. B. Molento Æ R. Prichard muscle paralysis of the parasite, respectively (Arena et al. Institute of Parasitology, McGill University, 1995; Dent et al. 1997; Ros Moreno et al. 1999; Feng et al. 21, 111 Lakeshore Rd., 2002). After treatment, these drugs are found mainly in Ste. Anne-de-Bellevue, Quebec, H9X 3V9, Canada liver, bile and fat (Chiu et al. 1990; Afzal et al. 1994). Drug combinations are being tested in clinical trials A. Lifschitz Æ J. Sallovitz Æ C. Lanusse (&) to combat tumour cells that express the multidrug Laboratorio de Farmacologıá, Facultad de Ciencias Veterinarias, resistance (MDR) gene, characterised by the increased Universidad Nacional del Centro, expression of a membrane transport monomeric protein, Campus Universitario (7000), P-glycoprotein (P-GP) (Rosenberg et al. 1997). P-GP is a Tandil, Argentina molecule of 170 kDa arranged as two homologous E-mail: [email protected] Fax: +54-2293-426667 halves, each containing six transmembrane regions and a nucleotide binding site (Gottesman and Pastan 1993). In Present address: M. B. Molento cancer cells, P-GP functions as an ATP-driven active Laborato´rio de Doenças Parasita´rias, Universidade Federal de Santa Maria, efflux pump for many unrelated cytotoxic drugs, Pre´dio 44, Camobi, Santa Maria RS, Brazil reducing the intracellular drug concentration by 122 increasing drug efflux (Sharon et al. 1995). P-GP also Experimental design participates in the mechanism of active secretion of different molecules from the bloodstream to the gas- Evaluation of verapamil side effects: dose adjustment trointestinal tract. Gianni et al. (1997) have indicated With the objective of measuring side effects after verapamil treat- that even small modifications to the dose of MDR ment alone in sheep, healthy animals were randomly allocated modulators can have profound effects on the plasma using a 4·4 Latin square design, with washout periods of 10 days. disposition of chemotherapeutic drugs in cancer pa- All four animals per treatment period received a 4-ml SC injection tients. In clinical trials, Kerr et al. (1986), reported a in the ventral region of 0 (placebo), 0.2, 2, 3, 5, 10, 15, and 20 mg/ kg verapamil. Clinical observations were made before treatment significant pharmacokinetic interaction when verapamil and for up to 5 days post treatment (PT). Hay, minerals and water and adriamycin were used in combined treatment. were available ad libitum. Verapamil was able to increase adriamycin peak plasma levels with a longer terminal half-life with no increase of Effects of verapamil on ivermectin and moxidectin kinetics adriamycin toxicity. Kantola et al. (1998) reported that verapamil increased the mean peak concentration in The pharmacokinetics of ivermectin or moxidectin alone at the ) serum of simvastatin by 2.6-fold and that the area under therapeutic dosage of 200 lgkg1 body weight or concomitantly the serum concentration-time curve increased 4.6-fold. with verapamil at 3 mg/kg ·3, at 12 h intervals, were investi- gated in sheep using a complete randomised block design. Sheep In a perfused rat liver model, excretion of vincristine were fed hay plus a ration made of mixed ground grains given into the bile and its selective inhibition by verapamil twice daily. Water was available ad libitum. The animals had provided indirect evidence for the contribution of P-GP their wool trimmed on the ventral part of the neck to facilitate to the biliary excretion of vincristine, while hepatic up- blood collection. The objective was to assess whether the pharmacokinetic behaviour of ivermectin or moxidectin changed take was not altered (Watanabe et al. 1992). Pouliot following drug combination treatment. Marriner et al. (1987) et al. (1996) demonstrated that ivermectin is a substrate demonstrated that when ivermectin is given orally the peak for P-GP. Xu et al. (1998) and Molento and Prichard plasma concentration is reached after 12–20 h PT. Verapamil has (1999) reported that verapamil increased the efficacy of a much shorter half-life in the animals, due to its extensive liver ivermectin and moxidectin when administered in com- first-pass metabolism (MacTavish and Sorkin 1989), than either ivermectin or moxidectin. Therefore, it was decided to administer bination with these compounds against a moxidectin- verapamil three times at 12 h interval in order to enhance the resistant strain of Haemonchus contortus. In veterinary possibility of seeing any effect on the pharmacokinetics of the medicine the use of P-GP modulators to modify the anthelmintics. pharmacokinetic behaviour of endectocide compounds Blood was collected in heparinised tubes at 0, 1, 2, 3, 8, 12, 18, 24, and 36 h and at 2, 3, 4, 5, 7, 9, 12, 15, 20, 25, and 30 days PT. in ruminant species has been reported (Lifschitz et al. Plasma was immediately separated by centrifugation at 3,000 g and 2002; Dupuy et al. 2003) In view of the fact that stored at )20C before analysis. Pharmacokinetic analysis of both verapamil increases the activity of these anthelmintics in macrocyclic lactones was performed using high performance liquid vivo, and that biliary and intestinal P-GP may play a chromatography (HPLC) with automated solid-phase extraction and fluorescence detection (Alvinerie et al. 1995; Lifschitz et al. role in the elimination of the macrocyclic lactones, it was 1999). Animals were monitored daily for any external clinical of interest to determine whether the MDR-reversing symptoms. Briefly, a 1 ml aliquot of plasma sample was combined agent verapamil, used in combination with ivermectin with 100 ll internal standard (abamectin 100 ng/ml) and then and moxidectin, could alter the pharmacokinetic profiles mixed with 1 ml acetonitrile. After mixing, the solvent-sample of these macrocyclic lactones in comparison with those mixture was centrifuged at 2,000 g and the supernatant obtained was placed on the appropriate rack of an Aspec XL autosampler of ivermectin and moxidectin given alone to sheep. (Supelco, Villiers le Bel, France) and automated solid-phase extraction was performed. The analytes were eluted with 1 ml methanol and concentrated to dryness under a stream of nitrogen. Reconstitution was done using 100 ll of a solution of N-methy- Materials and methods limidazole (Sigma) in acetonitrile (1:1). Derivatisation was initiated by adding 150 ll trifluoroacetic anhydride (Sigma) solution in Drugs acetonitrile (1:2). After completion of the reaction (<30 s), an aliquot (100 ll) of this solution was injected directly into the HPLC. All animals were treated with ivermectin (Ivomec; CDMV, Ste. Ivermectin and moxidectin plasma concentrations were determined Hyacinthe, PQ, Canada), or moxidectin (Cydectin, Fort Dodge using a Shimadzu 10A HPLC system (Shimadzu, Kyoto, Japan). Animal Health, Buenos Aires, Argentina) alone or in combination HPLC analysis was undertaken using a reverse phase C18 column with verapamil (Sigma, St. Louis, Mo.). The anthelmintics were (Phenomenex, 5 lm, 4.6 mm ·250 mm) kept in a column oven at administered orally (PO) and verapamil was diluted in saline 30C (Shimadzu) and a fluorescence detector (Spectrofluorometric solution prior to subcutaneous (SC) treatment. detector RF-10, Shimadzu), reading at an excitation wavelength of 365 nm and emission wavelength of 475 nm.

Animals Pharmacokinetic calculations Four 1-year-old sheep (30–40 kg) were used to assess side effects due to verapamil treatment. Separately, 32 1-year-old sheep The plasma concentration vs time curves obtained after each weighing between 33 and 43 kg were selected for the pharmacoki- treatment in each individual animal were fitted using PK Solutions netic evaluation. All animals were ear-tagged. For pharmacokinetic computer software (Ashland, Ohio). Pharmacokinetic parameters evaluation, animals were randomly divided in four groups of eight were determined using a model-independent method. The peak sheep each and allocated to open wooden pens with concrete floors, plasma concentration (Cmax) and time to peak plasma concentra- from prior to dosing until the end of the experiment. tion (Tmax) were read from the plotted concentration-time curve for 123 each animal. The terminal (elimination) half-life (T1/2 el) and 30 min. The animals that received verapamil at 3, 2, and absorption half-life (T1/2 ab) were calculated as ln 2/b and 2/kab, 0.2 mg/kg presented no clinical manifestations. There- respectively, where b is the terminal slope and kab, is the rapid slope obtained by feathering, which represents the first order absorption fore, the dosage of verapamil chosen for the following rate constant. Areas under the concentration versus time curves experiments was 3 mg/kg body weight SC. (AUC) were calculated by the trapezoidal rule (Gibaldi and Perrier 1992) and further extrapolated to infinity by dividing the last experimental concentration by the terminal slope (b). Statistical Effects of verapamil on ivermectin and moxidectin moment theory was applied to calculate the mean residence time (MRT) for ivermectin and moxidectin as follows: kinetics

MRT ¼AUMC=AUC The plasma concentrations of ivermectin and moxidec- where AUC is as defined previously and AUMC is the area tin obtained after their oral administration alone or co- under the curve of the product of time and drug concentration vs administration with verapamil are shown in Figs. 1 and time from zero to infinity (Giraldi and Perrier 1992). Treat- ment comparisons were made using Students t test, and proba- 2, respectively. For all drug regimes the anthelmintics bility under 0.05 (P<0.05) was considered to be statistically were detected in plasma at the first sampling point 1 h significant. PT in sheep. Moxidectin exhibited a significantly higher Cmax than ivermectin (17.2 vs 6.88 ng/ml), respectively. However, the time of peak concentration (0.79 and Results 0.86 h, respectively) was similar. The MRT of moxidectin was 6.2-fold greater than that of ivermectin and Evaluation of verapamil side effects: dose adjustment the AUC was 4.9-fold greater (Table 1). The combination ivermectin/verapamil caused sig- Verapamil given to animals at 20 mg/kg induced exter- nificant changes in the pharmacokinetic behaviour of nal manifestations that started 1.5 h PT. Symptoms in- ivermectin (Table 1). However, verapamil had no effect cluded an increase in respiratory frequency (160/min), on the kinetics of moxidectin (Table 1). The ivermectin two-step breathing, decrease in activity, loss of equilib- plus verapamil treatment resulted in higher ivermectin rium (difficulty in walking), and loss of appetite. Tem- plasma concentrations over the whole drug detection perature and ocular membrane colour were not changed period of 12–15 days compared with the ivermectin and the recovery of the animals to normal parameters alone group. The AUC of ivermectin was significantly occurred 6 h PT. External manifestations started 1 h higher (P=0.022) following co-administration (Fig. 3). after treatment of animals with verapamil at 15 and The Cmax of ivermectin was 83% higher (P=0.048) after 10 mg/kg. The clinical symptoms detected in sheep ivermectin plus verapamil treatment compared with varied from apathy, tremor, dizziness, and increased treatment with ivermectin alone (Fig. 3). Ivermectin respiratory rate to up to 140/min. Animals had difficulty concentration at 12 days PT was 57% higher following breathing and extended their necks to facilitate the the ivermectin plus verapamil treatment. The T1/2 el for process. Some manifestations deteriorated (respiratory ivermectin was longer (P<0.05) in the presence of ver- rates) by 2 h PT, but animals returned to normal by 3.5 apamil. Although the differences in T1/2 ab and Tmax and 4 h PT after 10 and 15 mg/kg, respectively. The between the drug combination and the ivermectin alone animals that received 5 mg/kg verapamil showed some treatment did not achieve statistical significance, they mild side-effects, such as apathy, 1.5 h PT lasting for tended to be higher in the presence of verapamil.

Fig. 1 Ivermectin plasma concentrations obtained after its oral administration (at time = 0) either alone (200 lg/ kg) or co-administered with verapamil (3 mg/kg ·3, at 12 h intervals) to sheep. Mean ±SEM (n=8). › Verapamil treatments 124

Fig. 2 Moxidectin plasma concentrations obtained after its oral administration (at time =0) either alone (200 lg/kg) or co-administered with verapamil (3 mg/kg ·3, at 12 h intervals) to sheep. Mean ±SEM (n=8). Inset Moxidectin concentrations (log scale) over a period of 4 days post-treatment (PT). › Verapamil treatments

Expression of P-GP is tissue-specific, with quite Discussion distinct levels of expression and function in different tissues. The class 3 gene (MDR2 in humans) is highly The differences in the MRT and AUC between moxi- expressed in the apical surface of the biliary canalicular dectin and ivermectin pharmacokinetic parameters membrane of hepatocytes facing an excretory compart- reflect the persistent efficacy demonstrated by moxidec- ment, functioning as a transport protein for many tin in the field (Rendell and Callinan 1996). The phar- products into bile. Although not fully clarified, macokinetic parameters MRT and AUC for moxidectin this MDR class, in contrast to class 1 or 2 does not after oral administration are in close agreement with confer MDR but its function can be blocked by the use those reported by Alvinerie et al. (1998). The values for of MDR modulators, such as the calcium channel Cmax, Tmax,andT1/2 el fall within the standard deviation blocker, verapamil. The knowledge that P-GP is ex- range reported by Alvinerie et al. (1998). The values of pressed in normal tissues and that MDR modulators Cmax, Tmax, AUC, and T1/2 el for ivermectin are very have access to it has raised many therapeutic possibili- similar to those reported by Marriner et al. (1987). ties. Thus, many sites for drug-drug interaction have been suggested, including absorption sites in the gastrointestinal tract, and the carrier-mediated transport Table 1 Pharmacokinetic parameters (mean ±SEM) for ivermectin (IVM) and moxidectin (MXD) obtained after their administration either across plasma membranes; e.g. hepatic or renal uptake, in the presence or in the absence of verapamil (VRP) to sheep (n=8 per secretion, or metabolism (Ito et al. 1998). group). T1/2ab Absorption half-life, Cmax peak plasma concentration, P-GP is expressed in liver, where it is found only at Tmax time to peak plasma concentration, AUCtotal area under the con- the apical pole of the canalicular membrane, where it is centration vs time curve extrapolated to infinity,MRT mean residence believed to be involved with transmembrane transport time, T el elimination half-life 1/2 (Bushman et al. 1992). Ruetz and Gros (1995), using Kinetic Treatmentsa yeast secretory vesicles stably expressing mdr2, tested parameters the translocation of phosphatidylcholine into bile in the IVM IVM+VRP MXD MXD+VRP presence or absence of mdr2 ()/), +/+). The increased mdr2-mediated translocation (+/+) was T1/2 ab (days) 0.29±0.05 0.34±0.09 0.20±0.04 0.17±0.02 Cmax (ng/ml) 6.88±0.59 12.6±2.67 17.2±2.61 12.3±1.63 abrogated by verapamil, demonstrating that mdr2 P- Tmax (days) 0.86±0.12 0.91±0.21 0.79±0.19 0.53±0.07 GP is a lipid transporter and functions as a flippase AUCtotal 19.3±2.13 29.7±3.71 94.9±16.2 62.3±10.2 and translocase moving lipids from one leaflet of the (ngÆday)1 ml)1) MRT (days) 2.85±0.32 3.21±0.20 17.7±2.56 19.7±4.24 membrane to the other (Ruetz and Gros 1994). As T1/2 el (days) 2.14±0.23 2.90±0.23 15.4±1.98 16.2±2.57 mentioned above, ivermectin is a substrate of P-GP. Thus, the transport of ivermectin into and out of the a Ivermectin and moxidectin were given orally at 200 lg/kg and verap- liver might occur via mdr2 P-GP, facilitating the pas- amil was given subcutaneously at 3 mg/kg ·3, at 12 h intervals. The sage of ivermectin into the bile. If that is so, an pharmacokinetic parameters Cmax, AUC and T1/2 el were significantly different between the ivermectin and ivermectin + verapamil treatment explanation for our data could be that verapamil is groups (P<0.05) altering ivermectin pharmacokinetics by decreasing its 125

compared with moxidectin (Lespine et at. 2003). However, pharmacokinetic modifications were obtained after the in vivo co-administration of moxidectin with P-GP substrate compounds. After co-administration of moxidectin with loperamide in cattle (Lifschitz et al. 2002), and with the flavonoid quercetin in lambs (Du- puy et al. 2003), the systemic availability of the antiparasite compound increased between 46% and 83%, respectively. Therefore, possible differences in the involvement of P-GP in the efflux of ivermectin and moxidectin need further evaluation. It is also unclear if the modulation of P-GP at the biliary/intestinal surface would have any consequences for increasing host toxicity to combinations involving MDR-reversing agents. In our studies, sheep only Fig. 3 Comparative mean (n=8) peak plasma concentrations expressed major signs of toxicity after verapamil was (Cmax), and area under the concentration vs time curves (AUC), for ivermectin after its oral administration either alone or co- given alone at 10 mg/kg or above. This could be administration with verapamil in sheep attributed to the vasodilatory action of verapamil. The combination of ivermectin or moxidectin with verapamil at 3 mg/kg ·3, at 12 h intervals, was not toxic to the biliary excretion and consequently decreasing drug animals. clearance. The presence of P-GP in the intestine con- Metabolic drug interactions have been reported and stitutes a barrier that limits the absorption of iver- the alteration of ivermectin bioavailability might have mectin administered orally (Kwei et al. 1999). been dependent on alterations in the rate of ivermectin Therefore, the higher Cmax (83%) obtained after the co- metabolism. CYP3A4 was found to be the main isoform administration of ivermectin with verapamil compared of the P450 enzyme responsible for ivermectin metabo- with treatment with ivermectin alone may reflect an lism (Zeng et al. 1998). This enzyme is responsible for the increase in the absorption of the antiparasite com- metabolism of many drugs and is expressed in liver and pound from the gastrointestinal tract. Moreover, P-GP in the intestine (Lown et al. 1993; Thummel et al. 1996). may play a relevant role in the intestinal elimination Other studies revealed the overlapping substrate/inhibi- process of different compounds. In rats, the amount of tor specificity of CYP3A4 and P-GP for verapamil ivermectin parent drug actively secreted in the small (Wacher et al. 1995), suggesting that the formation of the intestinal lumen was five times higher than that elimi- metabolite norverapamil is through CYP3A4 after nated in bile (Laffont et al. 2002). The increase of incubation with verapamil (Kroemer et al. 1993). The ivermectin absorption from the gastrointestinal lumen possibility of common regulatory factors for CYP3A4 and the decrease of biliary or intestinal ivermectin and P-GP proteins has been demonstrated by Schuetz elimination may contribute to the higher ivermectin et al. (1996), suggesting that verapamil is a good inhibitor availability measured as AUC (54%) obtained after co- for P-GP and for CYP3A4. Thus, drug-drug interaction administration with verapamil. based on metabolic inhibition involving CYP3A4 cannot There are at least two possible explanations for the be neglected. However, in view of the fact that most of lack of effect of verapamil on moxidectin pharmaco- the administered ivermectin is excreted unchanged, we kinetics. The first is that moxidectin is more rapidly believe that the inhibition of CYP3A4 by verapamil distributed to fat than is ivermectin, from where it is would play only a minor role in the altered ivermectin slowly eliminated. The effects of verapamil may be only pharmacokinetic profile when co-administered with transient, due to its much shorter half-life. A second verapamil. It seems likely that the principal cause of possible explanation is that moxidectin may be a verapamil-mediated alteration of ivermectin pharmaco- weaker P-GP substrate than ivermectin, as suggested kinetics may be based on competition for the P-GP by preliminary experiments (J.F. Pouliot, E. Georges transport mechanism. In conclusion, important changes and R. Prichard, unpublished data). These possibilities in the pharmacokinetics of ivermectin were obtained could explain the lack of effect of verapamil on the after its co-administration with a non-toxic dose of elimination of moxidectin, and may also account for verapamil in sheep. the much longer residence time of moxidectin compared with ivermectin. Under normal circumstances, Acknowledgements This work was partially supported by the ivermectin may be actively excreted by the action of P- Consejo Nacional de Investigaciones Cientı´ficas y Tećnicas (Con- GP in the hepatic-biliary system, whereas moxidectin icet, Argentina) and Secretarıá de Ciencia y Tećnica, Universidad may be little affected by this efflux mechanism, leading Nacional del Centro (Argentina), the Natural Sciences and Engi- neering Research Council of Canada and Fonds pour la Formation to its long residence time. Using genetically engineered des Chercheurs et laide a` la Recherche (Quebec). M. Molento was cell lines (LLCPK1) that express murine P-GP, iver- the recipient of the Blair Postgraduate Fellowship, McGill mectin was a stronger inhibitor of rhodamine efflux University. 126

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