FULL PAPER Toxicology Central and Peripheral Neurotoxic Effects of Ivermectin in Rats Saša M TRAILOVIĆ1) and Jelena Trailović NEDELJKOVIĆ2) 1)Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, University of Belgrade, Bulevar oslobodjenja 18, Belgrade 11000 and 2)Faculty of Veterinary Medicine, Department of Nutrition, University of Belgrade, Bulevar oslobodjenja 18, Belgrade 11000, Serbia (Received 27 September 2010/Accepted 6 December 2010/Published online in J-STAGE 20 December 2010) ABSTRACT. Ivermectin is considered a very safe drug; however, there are reports of toxic effects in particularly sensitive populations or due to accidental overdose. The aim of this study was (1) to further characterize the central and peripheral toxic effects of ivermectin in animals and (2) to determine possible therapeutic strategies for use in cases of ivermectin poisoning. We tested the effects of exper- imental doses of ivermectin previously reported to cause various intensities of CNS depression. However, in our study, ivermectin at 2.5, 5.0 and 7.5 mg/kg i.v. did not produce visible CNS depression in rats and 10 mg/kg resulted in sleepiness and staggering 10 to 40 min after application, while a dose of 15 mg/kg caused CNS depression very similar to general anesthesia. Ivermectin dose-dependently potentiates thiopentone-induced sleeping time in rats. Flumazenil (0.2 mg/kg), the benzodiazepine antagonist, did not affect the action of thiopentone; however, it significantly reduced sleeping time in rats treated with a combination of ivermectin (10 mg/kg) and thiopen- tone (25 mg/kg; from 189.86 45.28 min to 83.13 32.22 min; mean SD). Ivermectin causes an increase in the tonus (EC50=50.18 M) and contraction amplitude (EC50=59.32 M) of isolated guinea pig ileum, very similar to GABA, but without the initial relaxation period. These effects are dose-dependent and sensitive to atropine. Our results confirm the central and peripheral GABAergic properties of ivermectin in mammals and also indicate involvement of the cholinergic system in its toxicity. In addition, the results suggest that flumazenil and atropine have potential clinical roles in the treatment of ivermectin toxicity. KEY WORDS: flumazenil, GABA, isolated ileum, ivermectin, thiopentone. J. Vet. Med. Sci. 73(5): 591–599, 2011 Ivermectin (22,23-dihydroavermectin B1a) is a macrocy- capillary barrier easily and enters the brain tissue thereafter. clic lactone widely used as an antiparasitic agent in veteri- Previously, it has been demonstrated that ivermectin pro- nary and human medicine. Ivermectin belongs to the duces a direct agonistic action on the GABAA receptor. avermectins and is primarily isolated from fermentation There is also an opinion that avermectins in general act at products of the fungi Streptomyces avermitilis [6, 35]. The sites recognized by benzodiazepines on the GABA receptor- target of its antiparasitic action is an ivermectin-sensitive ionophore complex chloride channel. We have demon- glutamate gated Cl– channel receptor (GluCLR) that exists strated previously that ivermectin has important anticonvul- only in a number of invertebrates and the GABAA receptor. sive properties in lidocaine and strychnine-induced However, ivermectin and other avermectins can sometimes convulsions, although these have different mechanisms produce side effects in hosts. These effects can be related to [41]. Other authors have shown similar anticonvulsant GABAergic neurotransmission. Ivermectin is thought to actions of ivermectin in pentylenetetrazole-induced seizures promote the release of GABA and/or acts as a GABA recep- [12]. Furthermore, some authors reported transient gas- tor agonist, thus raising the GABA-induced chloride con- trointestinal upset either in humans or in animals poisoned ductance [36, 37, 44]. by ivermectin, which may imply an action on neuronal tis- The most dominant clinical symptoms of ivermectin poi- sue in the stomach and gut [3]. soning in dogs and cats [24], sheep [5], pigs [38], horses Ivermectin in the mammalian central nervous system [19] and other domestic and wild animals are CNS depres- interacts with at least three targets, a GABA-dependent sion and sometimes coma, frequently resulting in death. chloride channel, a glycine-dependent chloride channel and There is no evidence of ivermectin neurotoxicity in humans, a voltage-dependent chloride channel [1, 13, 15, 32]. who are mostly treated against filariasis. This can be Through these interactions, ivermectin realizes its central explained by the high efficacy of p-glycoprotein, a trans- depressive effect, depending on the concentration in the membrane protein highly conserved in the human popula- CNS. All these findings are mainly derived in vitro from tion [22] that plays a central role in limiting drug uptake electrophysiological investigation or ligand binding assays. into the brain [14]. In domestic animals, the depressive It is difficult to explain why proven in vitro antagonists of effect of ivermectin is common and may include more than different types of chloride channel (picrotoxin, DIDS, one mechanism. Certainly, ivermectin penetrates the brain strychnine, etc.) have no therapeutic value in treatment for poisoning with ivermectin [1, 39]. Alternatively, as we have *CORRESPONDENCE TO: TRAILOVIć, S. M., Department of Pharma- cology and Toxicology, Faculty of Veterinary Medicine, Uni- noted previously, there is substantial evidence that ivermec- versity of Belgrade, Bulevar oslobodjenja 18, Belgrade 11000, tin interacts with the binding sites of benzodiazepines on the Serbia. GABA receptor-ionophore complex chloride channel [25, e-mail : [email protected] 592 S. M. TRAILOVIĆ AND J. T. NEDELJKOVIĆ 45]. valve were removed. Gut contents were removed by flush- The aim of the study was to further characterize the cen- ing the lumen with saline solution (NaCl 0.9%), and the gut tral and peripheral toxic effects of ivermectin in mammals was placed in bubbled (95% O2/5% CO2) Krebs solution and to determine possible therapeutic strategies for use in containing (mM) NaCl 139.9, KCl 2.7, CaCl 1.8, MgCl2 o cases of ivermectin poisoning. Selection of the ivermectin 1.04, NaHCO3 11.9, NaH2PO4 0.4 and glucose 5.5. at 37 C. doses used in the study was based on the available literature Drugs and method of administration: Sigma Chemical on experimental doses able to cause CNS depression as well Co. (St. Louis, MO, U.S.A.) supplied ivermectin, GABA, as poisoning in different animal species [5, 12, 24]. We acetylcholine and atropine. Flumazenil (Anexate®) was were interested in whether ivermectin prolonged thiopen- obtained from Roche Pharma (Reinach, Switzerland), and tone-induced sleeping behaviors and whether this effect was thiopentone sodium (Trapanal® was obtained from Byk dose-dependent. Also, we chose to investigate the efficacy Gulden (Germany). Ivermectin was dissolved in propylene of flumazenil (clinically effective benzodiazepine-receptor glycol + glycerol mixture (60:40 v/v), thiopentone sodium antagonist) in neutralizing the central depressive action of was dissolved in water for injections and flumazenil (Anex- ivermectin in a animal model. The central depressive effect ate®) was administrated in the original formulation. All of ivermectin in rats was evaluated on the basis of duration drugs were dissolved immediately prior to application. of sleeping time after application of increasing doses of the All drugs were administered by slow intravenous injec- drug. The depressive effect on the CNS was also evaluated tion (via the lateral tail vein) using an intravenous cannula through the influence of ivermectin on the sleeping time in (OptivaTM 2, Johnson & Johnson, Arlington, TX, U.S.A.). rats caused by thiopentone. Changes in barbiturate-induced In the case of multiple applications, the time between 2 sleep time can be a useful tool for examining stimulatory or injections was 3 min, and total volumes did not exceed 0.1 inhibitory effects on the CNS, in particular for investigating ml/100 g body weight. influences on the GABAergic system. Barbiturates interact Experimental design with GABA receptors and modulate GABAergic effects, Investigation of ivermectin depressive effect on the rat while many hypnotic, antianxiety and anti-epilepsy drugs CNS: To study the potential depressive effect of ivermectin prolong barbiturate-induced sleeping time [30]. To eluci- on the CNS, rats were treated with increasing intravenous date the peripheral toxic action of ivermectin, we tested the doses of this drug. Animals were divided into 6 groups effects of high concentrations on the basal tonus and ampli- (n=15), one control group and 5 ivermectin-treated groups tude of isolated guinea pig ileum. We chose supratherapeu- (2.5, 5, 7.5, 10 and 15 mg/kg). Ivermectin, or solvent for tic doses of ivermectin to mimic situations where overdose control rats, was injected intravenously, whereby applica- or accidental ingestion happened [18, 26]. In the enteric tion always lasted for 30 sec. nervous system, there is a well-described direct link Thiopentone-induced sleeping time: To determine the between GABAergic and cholinergic function [40]; there- dose-dependent sleeping time caused by thiopentone, inves- fore, it was important to examine whether the effect of iver- tigations were done on a total of 105 rats divided in 7 equal mectin was sensitive to atropine, a broad-spectrum groups (n=15 per dose), one control and 6 thiopentone- muscarinic antagonist. treated groups (10, 15, 20, 25, 30 and