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wileyonlinelibrary.com/journal/jvp © 2019 John Wiley & Sons Ltd J vet Pharmacol Therap. 2019;42:654–659.1 | INTRODUCTION
In the pet trade, stressful life, poor caretaking, the presence of dif‐ ferent species, and a high number of animals in small living space suppress the immune response and increase the opportunity for parasites in animals. In addition, the widespread trade of pet tur‐ tles has increased the transmission of parasites between natural and exotic turtles (Hidalgo‐Vila et al., 2009; Rataj, Lindtner‐Knific, Vlahovic, Mavri, & Dovc, 2011). Nematodes, one of the most com‐ monly encountered parasite species in veterinary medicine because of their life cycle, are abundant in the gastrointestinal tract of turtles (Machin, 2015). The common nematode species in red‐eared slider turtles are Angusticaecum, Camallanus spp., Serpinema, Spiroxys,
Falcaustra, and Tachygonetria spp. (Demkowska‐Kutrzepa et al.,
2018). Parasitic nematodes may cause excessive weight loss, gas‐ trointestinal tract obstruction, gastritis, hepatic lipidosis, enteritis, ulceration, peritonitis, pancreatitis, and death in turtles (Chitty & Raftery, 2013; Hidalgo‐Vila et al., 2011; Martínez‐Silvestre, Guinea, & Pantchev, 2015).
Levamisole is a broad‐spectrum anthelmintic used in the treat‐ ment of pulmonary and gastrointestinal nematode infections in humans and animals (Sajid, Iqbal, Muhammad, & Iqbal, 2006). Its anthelmintic effect originates from the stimulation of sympathetic and parasympathetic ganglia in susceptible parasites, resulting in paralysis (Yadav & Singha, 2011). Furthermore, levamisole has an immunomodulatory effect in humans and animals (Symoens & Rosenthal, 1977). Although nematodes are abundant in turtles (Demkowska‐Kutrzepa et al., 2018), there are a limited number
Received: 30 November 2018
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Accepted: 4 March 2019 DOI: 10.1111/jvp.12763P H A R M A C O K I N E T I C R E P O R T
Pharmacokinetics of levamisole in the red‐eared slider turtles
(Trachemys scripta elegans)
Orhan Corum
1| Duygu Durna Corum
1| Orkun Atik
2| Feray Altan
3|
Ayse Er
4| Kamil Uney
41Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Kastamonu, Kastamonu, Turkey
2Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Afyon Kocatepe, Afyonkarahisar, Turkey
3Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Dicle, Diyarbakir, Turkey
4Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Selcuk, Konya, Turkey
Correspondence
Orhan Corum, Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Kastamonu, Kastamonu, Turkey.
Email: [email protected]
Abstract
The pharmacokinetics and bioavailability of levamisole were determined in red‐eared slider turtles after single intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration. Nine turtles received levamisole (10 mg/kg) by each route in a three‐ way crossover design with a washout period of 30 days. Blood samples were col‐ lected at time 0 (pretreatment), and at 0.25, 0.5, 1, 1.5, 3, 6, 9, 12, 18, 24, 36, and 48 hr after drug administration. Plasma levamisole concentrations were determined by a high‐performance liquid chromatography assay. Data were analyzed by noncom‐ partmental methods. The mean elimination half‐life was 5.00, 7.88, and 9.43 hr for IV, IM, and SC routes, respectively. The total clearance and volume of distribution at steady state for the IV route were 0.14 L hr−1 kg−1 and 0.81 L/kg, respectively. For the IM and SC routes, the peak plasma concentration was 9.63 and 10.51 μg/ml, re‐ spectively, with 0.5 hr of Tmax. The bioavailability was 93.03 and 115.25% for the IM and SC routes, respectively. The IM and SC route of levamisole, which showed the high bioavailability and long t1/2ʎz, can be recommended as an effective way for treat‐ ing nematodes in turtles.
K E Y W O R D S
of studies based on dose determination of anthelmintic drugs. Levamisole, fenbendazole, and ivermectin are recommended in the treatment of nematode infections in reptiles. Because iver‐ mectin is toxic in chelonians, its use is not recommended in this species (Machin, 2015). Levamisole and fenbendazole were found to have comparable efficacy against nematodes in Houston toads (Bianchi, Johnson, Howard, & Crump, 2014). However, concerns have been raised on the use of fenbendazole because it results in changes in the blood and biochemical parameters in Herman's tortoises (Neiffer, Lydick, Burks, & Doherty, 2005), causes mye‐ losuppression in turtles (Innis, 2008), and parasites have devel‐ oped resistance to it (Machin, 2015). Levamisole can be used as an antinematodal agent in turtles (Flanagan, 2015; Lafebre, Martelli, & Murray, 2004). Although literature review revealed pharmaco‐ kinetic studies on the use of levamisole in rabbits (García, Diez, Sierra, & Terán, 1994; Villanueva et al., 2003), sheep (Fernández, García, Sierra, Diez, & Terán, 1997), goats (Sahagún et al., 2000), broiler chickens (El‐Kholy, Kemppainen, Ravis, & Hoerr, 2006), and fish (Zanon, Cerozi, Silva, & Cyrino, 2013), no study was found on its use in reptiles, including turtles. Therefore, this study aimed to determine the pharmacokinetics and bioavailability of levamisole following intravenous (IV), intramuscular (IM), and subcutaneous (SC) administrations at a dose of 10 mg/kg in red‐eared slider turtles.
2 | MATERIALS AND METHODS
2.1 | Chemicals
Levamisole hydrochloride (≥99%) analytic standard was obtained from Sigma–Aldrich (St. Louis, Mo., USA). Acetonitrile (ACN), meth‐ anol (MeOH), disodium phosphate (Na2HPO4), and monosodium phosphate (NaH2PO4) were used at appropriate purities for high‐ pressure liquid chromatography (HPLC) and were supplied from Merck (Darmstadt, Germany). For the injection, a levamisole formu‐ lation (Levaject 10% injectable solution, Vetifarm, Istanbul, Turkey), which was diluted at the concentration of 20 mg/ml with sterile water, was used for drug administrations.
2.2 | Animals
Nine healthy red‐eared slider turtles of undetermined sex, with body weights ranging from 0.45 to 0.60 kg (mean 0.52 kg), which had not been administered any drugs in the past 2 months, supplied by a local pet shop (Konya/Turkey), were used in this research. Turtles were randomly and equally (n = 3) divided into three different 450 L aquariums with a water temperature of 22–24°C. A dry basking area was heated to 30°C by infrared lamp. Aquarium system consisted of custom‐built mechanical and biological filtration. Animals were fed a commercial feed daily (Sera Reptil Raffy P, GmbH, Heinsberg, Germany). During the research, the health status of the animals was evaluated according to physical examination, behavior, appe‐ tite, and biochemical parameters. The study was conducted after
a 15‐day acclimatization period. The study was approved by The Ethics Committee of the Faculty of Veterinary Medicine (University of Selcuk, Konya, Turkey).
2.3 | Experimental design and blood sampling
The study was performed in a crossover design (3 × 3 × 3) with at least a 30‐day washout period. At first, turtles received levamisole by IV (n = 3, external jugular vein), IM (n = 3, left deltoid muscle), and SC (n = 3, left front leg axillary area) routes at 10 mg/kg doses. After the washout period, treatments were alternated. Turtles were only removed from water at the blood collecting time. They were kept in the basking area throughout the collection of the blood sample (~1 min). Blood samples (~0.18 ml) were collected using an insulin injector washed previously with 0.05 ml of heparin sodium (Nevparin, Mustafa Nevzat, Istanbul, Turkey) from the dorsal cervical sinus (right and left) of each turtles be‐ fore drug administration (0 min) and at 0.25, 0.5, 1, 1.5, 3, 6, 9, 12, 18, 24, 36, and 48 hr after drug administration. Subsequently, blood sam‐ ples were centrifuged at 4,000 g for 10 min within 0.5 hr after blood collection. Plasma was harvested and stored at −70°C until analysis.
2.4 | Levamisole analysis
Levamisole concentration in turtle plasma was determined using the HPLC‐UV by modifying the previously defined method (Sari, Sun, Razzak, & Tucker, 2006). Briefly, 75 μl of turtle plasma samples was transferred to microcentrifuge tubes and 150 μl methanol was added. The mixture was vortexed for 30 s, and then samples were centrifuged at 8,000 g for 15 min. After centrifugation, the obtained clean supernatant was transferred into an autosampler vial and a 10 μl aliquot was injected to the HPLC system. The HPLC system (Shimadzu, Tokyo, Japan) consisted of a pump (LC‐20AT), an autosa‐ mpler (SIL 20A), a column oven (CTO 10A), a degasser (DGU‐14A), a SPD‐10A UV–VIS detector, and a system controller (CMB‐20A). Chromatographic separation was performed with a Gemini™ C18 col‐
umn (250 × 4.6 mm; internal diameter, 5 μm; Phenomenex, Torrance, CA), maintained 40°C. Levamisole was detected at a wavelength of 225 nm. The mobile phase consisted of 30% ACN and 70% phos‐ phate buffer (0.01 M, pH: 7.5) at a flow rate of 1 ml/min. Data analy‐ sis was performed using HP PC controlled LC solution software program (Shimadzu, Japan).
The HPLC method was validated by assessing selectivity, sensitiv‐ ity, recovery, precision, and accuracy. The stock solution of levamisole was prepared by dissolving standard levamisole powder in HPLC‐grade water to obtain a concentration of 1 mg/ml. This solution was used to prepare standards of 0.04–40 μg/ml in HPLC‐grade water or drug‐ free turtle plasma. The selectivity of the method was evaluated using blank plasma samples from 9 turtles. No interference was observed with biological compounds in plasma. Standard curves were linear over the range of 0.04–40 μg/ml (r2 > 0.9996). The limit of detection was
0.02 μg/ml based on a signal‐to‐noise ratio of 3:1, whereas the limit of quantitation was 0.04 μg/ml based on a signal‐to‐noise ratio of 6:1 and with the acceptable limit (coefficients of variation of <15% and bias
of ±15). The mean percentage recovery of levamisole in plasma was 97.8 ± 4.84%. The intra‐day and inter‐day assay coefficients of varia‐ tions and biases, which calculated by five replicate analyses of each level of three quality control samples at the concentration of 0.1, 1, and 10 μg/ml within 1 day or on five consecutive days, were <7.8% and ±6.0, respectively.
2.5 | Pharmacokinetic calculations
Pharmacokinetic parameters of levamisole following IV, IM, and SC administration were estimated by noncompartmental analy‐ sis using WinNonlin 6.1.0.173 software program (Pharsight Corporation, Scientific Consulting Inc., North Carolina, USA). Noncompartmental pharmacokinetic parameters calculated in‐ cluded terminal elimination half‐life (t1/2ʎz), mean residence time (MRT), area under the concentration vs. time curve (AUC), volume of distribution at steady state (Vdss), and total clearance (ClT). The peak plasma concentration (Cmax) and time to reach Cmax (Tmax) were determined by direct observation of data. The t1/2ʎz was cal‐ culated by ln 2/ʎz. The AUC was measured with linear/log method. Mean absorption time (MAT) was calculated as MRTIM,SC − MRTIV. Bioavailability (F) was determined using the following formula:
F = (AUCIM,SC/AUCIV) × 100.
2.6 | Statistical analyses
All values were shown as the mean ± SD. The AUCs were analyzed using one‐way analysis of variance and post hoc Duncan tests. Paired t test was used for the evaluation of differences between Cmax, Tmax, and bioavailability. The t1/2ʎz, MRT, and MAT were calculated as harmonic means and compared with the Wilcoxon's rank Sum test. All the statis‐ tical analyses were performed using SPSS 22.0 program (IBM Corp, Armonk, NY). p < 0.05 was accepted as statistically significant.
3 | RESULTS
Semi‐logarithmic plasma concentration–time curves, and plasma pharmacokinetic parameters of levamisole following IV, IM, and SC administrations at a dose of 10 mg/kg in red‐eared slider turtles are presented in Figure 1 and Table 1, respectively. Levamisole was de‐ tectable in the plasma for up to 36 hr following IV administration and for 48 hr following IM and SC administrations. The t1/2ʎz and MRT were significantly different between the three routes of administration. The AUC value obtained following SC administration was higher than that following IM administration. The mean bioavailability following IM and SC administrations was 93.03% and 115.25%, respectively.
4 | DISCUSSION
No local or systemic adverse effect was observed following IV, IM, and SC administrations of levamisole at a dose of 10 mg/kg
in red‐eared slider turtles. Levamisole had no adverse effect at a dose of 40 mg/kg in chickens (El‐Kholy et al., 2006) and at a dose of 5–10 mg/kg in ewes (Galtier, Escoula, Camguilhem, & Alvinerie, 1981). However, adverse effects, such as short‐term (3–10 min) hy‐ perexcitability, tremors, and deep respiratory movements, were ob‐ served following IV and IM administrations at a dose of 7.5 mg/kg in goats (Galtier et al., 1981; Sahagún et al., 2000).
In chelonian, because levamisole administration is recommended at a dose of 5–50 mg/kg (Dovč et al., 2002; Flanagan, 2015; Lafebre et al., 2004), a dose of 10 mg/kg was preferred in the present study. Oral, injectable, and pour‐on administrations of levamisole are rec‐ ommended in the treatment of lung and gastrointestinal nematode infections in cattle, sheep, goats, and pigs (Lanusse, Alvarez, Sallovitz, Mottier, & Bruni, 2007). Along with oral administration, injectable le‐ vamisole is also highly effective against gastrointestinal nematodes (Adediran & Uwalaka, 2015). However, it is rarely recommended be‐ cause of considerable differences in the bioavailability of drugs fol‐ lowing oral administration in reptiles (Anonymous, 2018). Therefore, IM and SC administration routes were preferred for levamisole in the present study. Because of the presence of the renal portal system in turtles, it is recommended that drugs be administered into the cranial half of the body (Gibbons, 2014). Therefore, IM and SC administra‐ tions of levamisole were performed to the left deltoid muscle and left front leg axillary area, respectively, in the present study.
The mean t1/2ʎz, Vdss, and ClT after IV administration of levamisole in red‐eared slider turtles were 5.0 hr, 0.81 L/kg, and 0.14 L hr−1 kg−1, re‐
spectively. The mean t1/2ʎz, Vdss, and ClT of levamisole ranged from 0.96 to 2.34 hr, 2.14–13.6 L/kg, and 2.36–4.24 L hr−1 kg−1 in some mamma‐
lian and avian species, respectively (El‐Kholy et al., 2006; Fernández et al., 1997; García, Diez, Sierra, & Terán, 1992; Sahagún et al., 2000; Villanueva et al., 2003). These values indicate that levamisole has long
t1/2ʎz, low Vdss, and slow ClT in red‐eared slider turtles. Lipophilic le‐
vamisole has a wide distribution volume. The binding activity of le‐ vamisole to plasma proteins in mammalian and avian species has been reported to range from 19% to 85% (Plante, Erian, & Petitclerc, 1981; Sahagún et al., 1997). However, the binding activity of levamisole to plasma proteins in reptiles, including turtles, is unknown. The reason for lower Vdss value of levamisole in turtles than in other species may
F I G U R E 1 Mean (±SD) semi‐logarithmic plasma concentration–
time curves of levamisole following intravenous (IV), intramuscular (IM), and subcutaneous (SC) administrations at the dose of 10 mg/ kg in red‐eared slider turtles (n = 9)
0.01 0.1 1 10 0 6 12 18 24 30 36 42 48 Concentr atio n (µg /ml ) Time (hr) IV IM SC
be the difference in its binding ratio to plasma proteins. Also, lymph contamination, which may cause the determination of high blood drug concentration, may cause for low Vdss value in turtle. Levamisole is in‐ tensively metabolized via oxidation and hydrolysis reactions in most species. Particularly, the liver and other tissues and organs play a role in levamisole metabolism (Villanueva et al., 2003). The amount of drug excreted unchanged in the urine following the administration of levamisole was 28% in dogs (Plante et al., 1981) and 5%–10% in pigs and goats (Nielsen & Rasmussen, 1983). The metabolites of levamisole detected in the urine also differ between the species (Galtier, Coche, & Alvinerie, 1983; Ho et al., 2009; Kouassi, Caille, Lery, Lariviere, & Vezina, 1986). The rate of metabolism has been reported to be lower in turtles than in mammals and birds (Divers, 2015). In addition, the rate of metabolism, blood flow through the kidney, and glomerular filtration rate in turtles vary depending on the ambient temperature (Anonymous, 2018; Crawford, 1991). The low elimination in turtles detected in the present study may be due to decreased blood flow through the kidney and decreased metabolism rate associated with low water temperature (22–24°C) and lower metabolism rate than the mammalian and avian species.
The mean t1/2ʎz and MRT0–∞ were longer following the extravas‐ cular administration of levamisole than following IV administration in turtles. The Tmax (0.5 hr) obtained following IM and SC adminis‐ trations of levamisole was similar to that previously reported in fish (0.5 hr, IM, 27°C, Zanon et al., 2013), whereas it was longer than that reported in some mammalian species (0.05–0.37 hr, IM‐SC, Galtier et al., 1981; García et al., 1994; Sahagún et al., 2000). Turtles are ectothermic animals, and their cardiac output and tissue perfusion rate vary depending on the ambient temperature—decreased ambi‐ ent temperature decreases the tissue perfusion rate (Kik & Mitchell, 2005), whereas increased ambient temperature increases the tis‐ sue perfusion rate (Hochscheid, Bentivegna, & Speakman, 2002). In the present study, the turtles were housed at 22–24°C, which may
have resulted in decreased cardiac output and tissue perfusion rate, thereby causing slower drug absorption and limited excretion. The prolonged t1/2ʎz and MRT0–∞ following extravascular administration in turtles may have been originated from prolonged Tmax associated with tissue perfusion. The mean t1/2ʎz and MRT0–∞ was longer follow‐ ing SC administration of levamisole than following IM administration in turtles. There were no significant difference Cmax and Tmax values following IM and SC administration of levamisole. Although there was no difference in Cmax between the groups, levamisole concentration was higher following SC administration at all sampling time points than following IM administration, which may have caused the pro‐ longed mean t1/2ʎz and MRT0–∞ following SC administration. Elevated
Cmax and prolonged t1/2ʎz following SC administration of levamisole in turtles may be considered as an advantage over IM administration.
The mean Cmax following IM and SC administrations of levami‐ sole at a dose of 10 mg/kg in turtles (9.63–10.51 μg/ml) was higher than that previously reported in sheep (3.43 μg/ml, 10 mg/kg, IM, Fernández, García, Sierra, Diez, & Terán, 1998), fish (12.79 μg/ml, 50 mg/kg, IM, Zanon et al., 2013), rabbits (5.39 μg/ml, 20 mg/kg, SC, García et al., 1994), and goats (1.17 μg/ml, 7.5 mg/kg, SC, Sahagún et al., 2000). This sizeable difference in Cmax of levamisole in turtles is unlikely to be attributable to differences in the assay for determin‐ ing levamisole concentrations. Lymph contamination is a common problem encountered during the collection of blood samples in che‐ lonians, which may have an effect on blood drug levels. The sampling sites for collection of a venous blood sample with the least lymph contamination in chelonians are the jugular vein and dorsal cervical sinus (Flanagan 2015; Norton, 2005). We preferred the dorsal cer‐ vical sinus (left and right) for serial blood collection instead of the jugular vein in pharmacokinetic study because of the small size of red‐eared slider turtles. Thus, lymph contamination to the sampling blood might be a reasonable explanation for this observation.
Parameter IV IM SC t1/2ʎz (hr) HM 5.00 ± 0.39 7.88 ± 0.50a 9.43 ± 0.75b AUC0‐∞ (hr*μg/ml) 75.38 ± 12.71 69.33 ± 12.75 84.93 ± 14.83c AUCextrap % 0.82 ± 0.28 1.69 ± 0.35 3.37 ± 0.33 MRT0–∞ (hr) HM 5.95 ± 0.51 9.74 ± 0.46a 11.85 ± 0.72b MAT (hr) HM – 3.69 ± 0.59 5.82 ± 0.56d ClT (L hr−1 kg−1) 0.14 ± 0.02 – – Vdss (L/kg) 0.81 ± 0.11 – – Tmax (hr) M – 0.5 0.5 Cmax (μg/ml) – 9.63 ± 1.30 10.51 ± 0.76 F % – 93.03 ± 17.71 115.25 ± 26.08e
aSignificantly different from IV and SC (p < 0.012). bSignificantly different from IV and IM (p < 0.012). c,d,eSignificantly different from IM with p values of < 0.017, 0.012, and 0.015, respectively. AUC: area under the concentration vs. time curve; AUCextrap %: area under the plasma concentration–time curve extrapolated from tlast to ∞ in % of the total AUC, ClT: total clearance; Cmax: peak plasma con‐ centration; F: absolute bioavailability; HM: harmonic mean; M: median; MAT: mean absorption time; MRT: mean residence time; t1/2ʎz: terminal elimination half‐life; Tmax: time to reach the peak plasma concentration; Vdss: volume of distribution at steady state.
TA B L E 1 Mean (±SD) plasma
pharmacokinetic parameters of levamisole following intravenous (IV), intramuscular (IM), and subcutaneous (SC)
administrations at the dose of 10 mg/kg in red‐eared slider turtles (n = 9)
Levamisole was entirely absorbed following IM and SC admin‐ istration with complete absolute bioavailabilities (F) of 93% and 115%, respectively. High absolute bioavailabilities have been pre‐ viously reported in rabbits [105%–134% (SC), García et al., 1994; ], sheep [77%–100% (IM), 79.2% (SC), Galtier et al., 1981; Fernández et al., 1998; ], and goats [100% (IM), 68%–77% (SC), Galtier et al., 1981; Sahagún et al., 2000; ]. In this study, the F of levamisole fol‐ lowing SC administration was >100% and higher than that after IM administration. Reasons for the F of >100% have been reported (Toutain & Bousquet‐Mélou, 2004). In this study, MATSC values of <MRTIV do not support a flip‐flop phenomenon. The inter‐occa‐ sion variability of clearance occurring with long washout period effect could be the cause of pharmacokinetic differences be‐ tween SC and IM administrations and the F of >100% following SC administration.
In conclusion, levamisole was well‐tolerated following IV, IM, and SC administrations at a dose of 10 mg/kg in turtles. The IM and SC administration of levamisole that showed high bioavailability and prolonged t1/2ʎz can be recommended as an effective method for the treatment of nematodes in turtles.
ACKNOWLEDGMENTS
This research did not receive any specific grant from funding agen‐ cies in the public, commercial, or not‐for‐profit sectors.
CONFLIC T OF INTEREST STATEMENT
The authors declare no conflicts of interest.
AUTHORS’ CONTRIBUTION
OC and KU contributed to conception, design, analysis, and acquisi‐ tion, drafted the manuscript, critically revised the manuscript, gave final approval, and agreed to be accountable for all aspects of work ensuring integrity and accuracy. DDC, OA, FA, and AE contributed to analysis, and agreed to be accountable for all aspects of work ensur‐ ing integrity and accuracy.
ORCID
Orhan Corum https://orcid.org/0000‐0003‐3168‐2510
Duygu Durna Corum https://orcid.org/0000‐0003‐1567‐991X
Orkun Atik https://orcid.org/0000‐0003‐2411‐7492
Feray Altan https://orcid.org/0000‐0002‐9017‐763X
Kamil Uney https://orcid.org/0000‐0002‐8674‐4873
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How to cite this article: Corum O, Corum DD, Atik O, Altan F,
Er A, Uney K. Pharmacokinetics of levamisole in the red‐ eared slider turtles (Trachemys scripta elegans). J vet Pharmacol
