A NEW APPROACH IN TREATMENT ACUTE IVERMECTIN TOXICITY IN MALE BALB-C MICE

Assist Prof. Lecturer Lecturer 1 Dept of Phys, Bioch and Pharma, Coll of Vet Med, University of Baghdad, Iraq 2 Dept of Vet Public Health, Coll of Vet Med, University of Baghdad, Ira 3 Dept of phys and Pharmacology,Coll of Vet Med ,Al-Qasim Green University, Babylon-Iraq Email:fab20062003@yahoo.com, falah@covm.uobaghdad.edu.iq ABSTRACT This study was conducted to investigate the potential therapeutic of flumazenil and neostigmine in single and combination in Balb-C mice subjected to ivermectin acute oral toxicity. Ivermectin-poisoning dose was considered the half dose of LD50 that computed in current study as 20.9 mg/kg BW. A total of 24 male Blub-C mice were divided randomly into four equal treatment groups and administered ivermectin-poisoning dose (10 mg/kg BW) and treated as follows: control group (C), treated orally with distilled water; neostigmine-treated group (NT), treated i.p. with 60 μg/kg BW of neostigmine; flumazenil-treated group (FT), treated i.p. with 7 μg/kg BW of flumazenil; and combination-treated group (CT), treated i.p. with 30 μg/kg BW of neostigmine plus 3.5 μg/kg BW of flumazenil. Oral administration of ivermectin at poisoning dose caused grooming, lethargy, depression, recumbency, with no mortalities recorded. The toxic signs of animals in FT and NT groups were disappeared within 1.23 and 1.98 h, respectively, comparing to animals in CT and C groups, in which the recovery time was prolonged to 40.62 and 4.52 h, respectively. Overall, both flumazenil and neostigmine medicines have the potential to overcome ivermectin acute poisoning in Bulb-C mice, with flumazenil being more efficient. However, the combination of both medicines can cause an adverse prognosis.


INTRODUCTION
Ivermectin is a macrocyclic lactone, broadspectrum antiparasitic (18,14). It has been evolved over the last three decades from being a veterinary "blockbuster" drug to a panacea for nematodal infestation and ectoparasitic diseases in humans (5), and widely used as a drug for the treatment of parasitic infection in laboratory rodents (16). Ivermectin as insecticide mediates its activity through potentiation gamma aminobutyric acid (GABA) neural and neuromuscular transmission, while mammals are relatively resistant to ivermectin poisoning as they have only central GABA-ergic synapses protected by the blood-brain barrier. However, at high doses, ivermectin may penetrate the bloodbrain barrier. It was reported that the peak of the ivermectin level in the brain reaches between 2-4 hs after administration. In mammalian species, CNS depression and ataxia are the most observed symptoms seen as a result of inhibitory GABA-ergic synapses (8). Ivermectin augments the release of GABA at presynaptic neurons. It is absorbed up to 95% after oral administration in simple stomached animals (17). Acute toxicity investigated in dogs received 40 mg/kg BW orally and death due to respiratory depression (7). Likewise, dogs administered 2 mg/kg BW showed tremors, ataxia, anorexia, and developed dehydrated (17). Kittens received 7.5 mg/kg BW, which is corresponding to 125 fold of recommended therapeutic dose, developed ataxia, salivation, and comatose within 24 hs. These animals were treated by 25 µg neostigmine methyl sulfate plus 5% dextrose saline (20 ml) I.V. showed transient improvement but died after 12 h (8). In a certain case of calf administered a bolus contained 100 mg ivermectin, developed various clinical signs included mydriasis, absence of pupillary and menace reflex, sticky salivation, normal body temperature, tachycardia, tachypnoea, dull, depressed, sudden fall with tremors, abnormal posture and gait. The animals were treated by activated charcoal (2g/kg BW PO TID), Diazepam (0.5 mg/kg bid) up to the disappearance of clinical signs, and aggressive fluid therapy was given for three days. Supportive treatment included intramuscular injection of theophylline (6.6mg/kg BW), vitamin B-complex with liver extract (3 mL), chlorpheniramine maleate (Avil) at 0.5 mg/kg BW daily for five days. A slight improvement was seen in calf after three days and complete recovery after five days of treatment (19). The main concern is the neurotoxicity, which in most mammalian species may manifest as central nervous system depression, and consequent ataxia as might be expected from potentiating of inhibitory GABA-ergic synapses (2,15). Flumazenil, an imidazobenzodiazepine, has a very rapid onset of action after parenteral administration. The affinity of flumazenil is between the α and γ2 subunits of the GABA A receptor, close to the benzodiazepine binding site (13). The predominant receptor composed of α1 mediates sedation and muscle relaxation, those with α2 or α3 anxiolysis and anticonvulsant effects and the extrasynaptic receptor with α5 amnesia (1). Neostigmine is an anticholinesterase, and this allows ACh to persist for a longer period enhancing transmission across neuromuscular junctions and causes muscle contraction (10). Due to accidental and improper use of ivermectin in both human and animals (8), a wide range of different cases of ivermectin toxicity have been occurred. As a result, different strategies to treat ivermectin toxicity have been developed. However, some of them are not fully effective, and developing a new approach to overcome ivermectin acute toxicity is still in need. Therefore, the objective of this study was to design another strategy to treat ivermectin acute toxicity in mice and to establish its effectiveness.

MATERIALS AND METHODS Animals and management
The other 24 mice were used in the acute toxicity study of ivermectin. The study carried out after approval of the Scientific Committee in the Department of Physiology, Biochemistry, and Pharmacology, College of Veterinary Medicine, University of Baghdad in accordance with ethical standards of animal welfare. A total of thirty male Balb-C mice (2-3 months) weighting 20-30 g were raised and bred for one month before beginning the study for acclimatization in the animal house of College of Veterinary Medicine, University of Baghdad. Tap water and standard pellet diet were provided ad libitum. Six of them were used for determining the LD 50 of ivermectin by the Up and Down method (6). Medicines and administration methods : All medicines used in this study were purchased from suppliers as indicated for each one: Kepromic® oral ivermectin (KEPRO B.V., Holand), neostigmine (Hameln phamacenticals GmbH, Germany), and flumazenil (Sandoz, Canada). Dosage rates of all medicines administered to mice by different routes were achieved by fitting the concentrations for getting a dose volume of 0.1 mL/10 g BW of mice. All medicines were dissolved in distilled water.

Experimental design
To induce ivermectin toxicity in Bulb-C mice, the half dose of LD 50 was chosen as the toxic dose. LD 50 was estimated using 6 animals by the Up and Down method as described previously (6). The other 24 mice were subjected for conduction treatment of acute toxicity of ivermectin. They were divided equally into four groups and administered single ivermectin-poisoning dose at 10 mg/kg BW (half dose of LD 50 as estimated in the current study) and treated as follows: Control group (C), treated orally with distilled water through gastric gavage; neostigmine-treated group (NT), treated intraperitoneally with neostigmine at 60 µg/kg BW; flumazeniltreated group (FT), treated intraperitoneally with flumazenil at 7 µg/kg BW; and combination-treated group (CT), treated intraperitoneally with neostigmine at 30 µg/kg BW plus flumazenil at 3.5 µg/kg BW. Treatment with medicines started as soon as the signs of ivermectin toxicity had risen. The number of cured and dead animals in each group and time of recovery from poisoning were recorded and considered as criteria of treatment effectiveness. Statistical analysis Data of time recovery were analyzed as one way-ANOVA using the GLM procedure of SPSS software version 22.00 (IBM SPSS Inc., Chicago, IL, USA). Means were separated using LSD at P<0.05. Results presented as Mean±SEM. Individual animals were considered the experimental unit (11).

RESULTS AND DISCUSSION
The acute toxicity study found that the oral LD 50 of ivermectin as estimated by the Up and Down method is 20.9 mg/kg BW in male Balb-C mice for 24 h ( Table 1). The ivermectin is considered an extremely toxic agent with grade 5 according to the classification of toxicity rating which depends on LD 50 value (12). This outcome means that at approximate teaspoonful of ivermectin orally may cause lethality in an adult human. The signs exhibited by morbid mice are listed in Table 2. These signs have nearly resembled previous acute studies conducted in mice, rats, rabbits, dogs, and monkeys. The typical signs of acute ivermectin toxicity were all attributed to effects on the central nervous system. These signs were most severe in CF-1 mice, which exhibited ataxia, bradypnoea, and tremors. The death occurred from approximately one to six h after treatment as reported by (24), however, the death in the present study occurred between 6-9 h. Also, various clinical signs of ivermectin poisoning have been reported in human (9), in which some of these signs were compatible with what we found such as increased salivation, diarrhoea, difficult breathing, muscle fasciculation, drooping of lips, bilateral mydriasis, depression, ataxia, recumbency, reduced pupillary reflex, absent menace reflex, rarely encephalopathy, and death. Serious neurological adverse events have been reported from large scale community-based ivermectin treatment campaigns against onchocerciasis volvulus in Africa (4). In our not published work, we also observed bradypnoea and mydriasis in local Iraqi rabbits exposed subcutaneously to a single dose (33.7 mg/kg BW) of ivermectin that is resembled the half LD 50 we estimated in this animal species. This draws attention that ivermectin poisoning is variable in different animal species and route of administration.

All doses are in mg/kg BW
The result of current study showed that oral administration of ivermectin at poisoning dose, which was considered as the half dose of LD 50 calculated, caused grooming, lethargy, depression, recumbency, and with no mortalities recorded. These acute toxic signs were relieved within 1.23±0.01 h in animals of the FT group, which treated with flumazenil at 7 µg/kg BW. This reduction in time recovery was significantly (P<0.05) lesser than the ones recorded in animals of C (4.52 ±0.10 h, treated with distilled water), NT (1.98 ± 0.15 h, treated with 60 µg/kg BW neostigmine), and CT (40.25±2.78 h, treated with 30 µg/kg BW of neostigmine plus 3.5 µg/kg BW of flumazenil) groups ( Figure 1). These findings nearly resemble the result of (25) who found that flumazenil at 0.2 mg/kg BW significantly reduced the sleeping time in rats treated with a combination of ivermectin (10 mg/kg BW) and thiopentone (25 mg/kg BW). However, we found that flumazenil at 7µg/kg BW, which is approximately equal to 0.035 of the dose used by Trailović and Nedeljković (25), was able to relieve the acute toxic signs of ivermectin. The difference between the dose amounts may be due to species variation, route of administration, criteria considered as an indication of recovery, and the agents used to induce acute toxicity. Ivermectin is thought to promote the release of GABA and/or acts as a GABA receptor agonist, thus raising the GABA-induced chloride conductance which results in hyperpolarization of the neuron (22,23, and 26). We believe that as flumazenil is GABA antagonists, it can inhibit the action of GABA and consequently counteract the toxic effect of ivermectin. In general, these drugs (GABA antagonists) produce a convulsion effect, which is confirmed with the appearance of convulsion for short period (disappeared within minutes), in the mice after administration flumazenil. The animals in NT group (treated with 60 µg/kg BW neostigmine intraperitoneally) showed a non-significant decrease in time needed to recover the acute toxic signs of ivermectin (1.98±0.15 h) in comparison with time recovery recorded in the animals of C group (4.52±0.10 h).
Neostigmine is an irreversible inhibitor of acetylcholinesterase (AChE) (21) since neostigmine stops cholinesterase from breaking down acetylcholine at the junctions between the nerves and the muscle cells. This prolongs the action of acetylcholine at the nerve endings and gives it more chance to act on the remaining acetylcholine receptors. Acetylcholine is known to cause a slow depolarization by blocking a tonically-active K + current, which increases neuronal excitability and may opposite the hyperpolarization status due to toxic dose of ivermectin which is exerting its effect through GABA receptor agonist. However, due to the presence of quaternary nitrogen in its structure, neostigmine is not simply crossing the blood-brain barrier and the crossing can occur only in exceptional conditions (20). The animals in CT group (treated simultaneously with both 30 µg/kg BW neostigmine and 3.5 µg/kg BW flumazenil) showed a significant increase (P<0.05) in time recovery (40.25±2.78 h) in comparison with the time recovery recorded in animals of both FT and NT groups, as well as the C group ( Figure 1). The poisoned mice showed cholinergic toxicity signs such as convulsion, tremors, constriction of pupils, and paralysis. In addition, two mice were dead within 10 h after receiving the combination of these two agents. We hypothesized that the synergistic effect of flumazenil and neostigmine exacerbated the acute toxicity of ivermectin, which is due to the exaggerated effect of accumulated acetylcholine postsynaptic due to neostigmine action, while flumazenil antagonizes the GABA receptors. We could conclude that both flumazenil and neostigmine medicines have the potential to overcome ivermectin acute poisoning in Balb-C mice, but flumazenil is more efficient one, while the combination of them have a bad prognosis.