Comparative study of mechanism of antiparasitic and toxic action of gabaergic and cholinergic antihelmintics

Abstract

GABAergic and cholinergic systems in the neuromuscular system of the parasitic nematodes are the main target of action for anthelmintic drugs. In the nematode cholinergic system pharmacological importance has primarily nicotinic-acetylcholine receptor (nAChR). On the other hand, the GABA-receptor was originally identified as the main site of action for the Avermectins and Milbemicins. However, later was shown that these drugs work on a new, previously never described glutamate-gated chloride ion channel, located in the nematode pharynx. The most important problems that are endangering the success of antiparasitic therapy are the development of resistance in the parasites and often occurrences of the toxic effects of antiparasitic drugs in host. In the order to elucidate the mechanisms of action of GABAergičkih and cholinergic anthelmintic, we examined the pharmacological characteristics of the representatives of these two groups of drugs in the isolated nerve-muscle preparation of large pig nematode Ascaris suum. Also, it was important to examine comparatively differences in the effects of cholinergic and GABAergic anthelmintic on the corresponding receptors in mammals (investigations were performed on isolated rat diaphragm and ileum), and thus to analyze the mechanisms of their adverse effects. In presented investigations we measured the effects of contraction or relaxation on isolated nematode or mammalian preparations and analyzed the results by using appropriate statistical methods (non-linear regression, ANOVA, t-test). Based on the results obtained in our investigations the following conclusions may be presented: (1) Agonists of L, N and B type of nematode nicotinic acetylcholine receptor (nAChR) studied on a model of neuromuscular preparations of A. suum, showed variable efficacy. Highest efficiency in the first group of tested agonists has pyrantel (agonist of L-type nAChR, EC50=0.010 μM, Emax=2.5g), then bephenium (agonist of B-type nAChR, EC50=0.37μM, Emax=2.7g) and at the end acetylcholine (an endogenous neurotransmitter, agonists of all three types of receptors L, N and B, EC50=6.12-6.45μM, Emax=1.71-2.07g). Highest efficiency in the second group of nicotinic receptor agonists has been demonstrated by tribendimidine (probably agonist of L-type nAChR, EC50=0.064μM, Emax=1.29g), followed by levamisole (agonist of L-type nAChR, EC50=0.34μM, Emax=0.68g) and at the end nicotine (an agonist of N-type nAChR, EC50=4.99μM, Emax=1.07g). (2) Nicotinic acetylcholine receptor of A. suum, that acting tribendimidine and nicotine, exhibit characteristics of both types of mammalian nAChRs: a) the characteristics of mammalian muscle type of nAChR, because it is sensitive primarily to pancuronium but also to tubocurarine, and b) the characteristics of neuronal nAChRs of mammals, because it is sensitive primarily to mecamylamine but also to hexamethonium. (3) GABA induced a dose-dependent relaxation of A. suum neuromuscular preparation, the value of the mean EC50 was 7.40μM, while piperazine causes relaxation with EC50 of 331μM. Receptor through which GABA and piperazine exhibit relaxation of A. suum is different from mammalian GABAA receptor, because it is insensitive to bicuculine (specific mammalian GABAA receptor antagonist). (4) Ivermectin and moxidectin by themselves do not lead to the relaxation of neuromuscular preparation of A. suum. Furthermore, ivermectin does not potentiate the relaxation caused by GABA. Ivermectin induces relaxation only when is administered after GABA or in the neuromuscular preparations of A. suum with high amplitude of spontaneous activity (spontaneous contraction and relaxation about 0.5g). Therefore, ivermectin for its action requires prior activation of GABA-dependent chloride channels. (5) Carvacrol 100 and 300μM, causes long-term relaxation of neuromuscular preparation of A. suum. Carvacrol significantly potentiates relaxation caused by GABA and significantly inhibits the dose-dependent contractile effects of ACh, shifting the EC50 value from control 5.22μM to the 13.88μM and 22.72μM, respectively. Carvacrol reduced the maximum contractile effect of ACh from control 3.1g to the 2.50g and 1.96 g, respectively. Therefore, this is probably a classical non-competitive physiological antagonism, which carvacrol manifests by itself in relation to ACh. (6) Neither one combination of different parameters of Electrical Field Stimulation (EFS) (10Hz to 100Hz, 0.01-1.0ms, 5 to 30V, from 2.0 sec. to continuous stimulation), do not causes indirect stimulation of the A. suum neuromuscular preparation (contractions are insensitive to mecamylamine)...

Gabaergički i holinergički sistem u neuro-mišićnom sistemu parazitskih nematoda su glavna ciljna mesta delovanja antihelmintika. U okviru holinergičkog sistema nematoda, farmakološki značaj ima pre svega nikotinski-acetilholinski receptor (nAChR). S druge strane, GABA-receptor parazitskih nematoda je prvobitno označen kao glavno mesto dejstva avermektina i milbemicina. Međutim, vremenom se pokazalo da ovi lekovi deluju i na jedan potpuno nov, do tada neopisani glutamat-zavisni hloridni jonski kanal u farinksu nematoda. Pored toga, značajno je da neki aktivni principi etarskih ulja imaju dokazana antiparazitska svojstva, i da postoje indicije da deluju upravo preko ova dva receptorska sistema. Osnovni problemi koji danas ugrožavaju antiparazitsku terapiju su razvoj rezistencije i često ispoljavanje toksičnih efekata antiparazitskih lekova. Da bi se bolje razumeli mehanizmi dejstva gabaergičkih i holinergičkih antihelmintika ispitali smo farmakološke karakteristike predstavnika ove dve grupe lekova na neuro-mišićnom preparatu velike nematode svinja Ascaris suum. Takođe, značajno je bilo komparativno ispitati razlike u dejstvu gabaergičkih i holinergičkih antihelmintika na odgovarajuće receptore sisara (ispitivanja su izvršena na izolovanoj dijafragmi i ileumu pacova) i na taj način analizirati mehanizme njihovih neželjenih efekata. U našim istražvanjima mereni su efekti kontrakcije ili relaksacije izolovanih preparata i odgovarajućim statističkim metodama obrađivani dobijeni rezultati (nelinearna regresija, ANOVA, t-tets). Na osnovu rezultata dobijenih u ispitivanjima doneti su sledeći zaključci: (1) Agonisti L, N i B tipa nikotinskog-acetilholinskog receptora (nAChR) nematoda, ispitivani na modelu neuro-mišićnog preparata A. suum, ispoljili su različitu efikasnost. Najvišu efikasnost u prvoj grupi ispitivanih agonista, ispoljio je pirantel (agonista L-tipa nikotinskog receptora EC50=0.010μM, Emax=2.5g), zatim befinijum (agonista B-tipa nikotinskog receptora EC50=0.37μM, Emax=2.7g) i na kraju acetilholin (endogeni neurotransmiter), agonista sva tri tipa L, N i B nikotinskog receptora (EC50=6.12–6.45μM, Emax=1.71–2.07g). Najvišu efikasnost u drugoj grupi ispitivanih agonista nikotinskog receptora ispoljio je tribendimidin (najverovatnije agonista L-tipa nikotinskog receptora, EC50=0.064μM, Emax=1.29g), zatim levamizol (agonista L-tipa nikotinskog receptora, EC50=0.34μM, Emax=0.68g) i na kraju nikotin (agonista N-tipa nikotinskog receptora, EC50=4.99μM, Emax=1.07g). (2) Nikotinski-acetilholinski receptor A. suum, na koji deluju tribendimidin i nikotin ispoljava osobine oba tipa nAChR sisara: a) karakteristike mišićnog tipa nAChR sisara, jer je osetljiv pre svega na pankuronijum ali i na tubokurarin; b) karakteristike neuronskog tipa nAChR sisara, jer je osetljiv pre svega na mekamilamin ali i na heksametonijum. (3) GABA izaziva dozno-zavisnu relaksaciju neuro-mišićnog preparata A.suum, sa vrednošću srednje EC50 od 7.40μM, dok piperazin relaksaciju izaziva sa EC50 od 331μM. Receptor preko koga GABA i piperazin ostvaruju relaksaciju A. suum različit je od GABA receptora sisara jer je neosetljiv na bikukulin (specifični antagonista GABAA receptora). (4) Ivermektin i moksidektin sami po sebi ne dovode do relaksacije neuro-mišićnog preparata A. suum. Ivermektin ne potencira relaksaciju koju izaziva GABA. Međutim, ivermektin dovodi do relaksacije samo kada je aplikovan posle GABA ili na neuro-mišićnim preparatima A. suum sa velikom amplitudom spontane aktivnosti (spontane kontrakcije i relaksacije od oko 0.5g). Ivermektin za svoje dejstvo najverovatnije zahteva prethodnu aktivaciju GABA-zavisnog hloridnog kanala. (5) Karvakrol u koncentracijama od 100 i 300μM, izaziva dugotrajnu relaksaciju neuro-mišićnog preparata A. suum. Karvakrol signifikantno potencira relaksaciju koju izaziva GABA i signifikantno inhibiše dozno-zavisne kontrakcije izazvane sa ACh, pomerajuću vrednost EC50 ACh sa kontrolnih 5.22μM, na 13.88μM i 22.72μM. Karvakrol smanjuje maksimalni kontraktilni efekat ACh sa kontrolnih 3.1g na 2.50g i 1.96g. Ovde se verovatno radi o klasičnom nekompetitivnom fiziološkom antagonizmu, koji karvakrol ispoljava u odnosu na ACh. (6) Ni jedna kombinacija različitih parametara električne poljne stimulacije (EFS) (10 do 100Hz, 0.01-1.0ms, 5 do 30V, od 2.0s do kontinuirane stimulacije), ne izaziva indirektnu stimulaciju neuro-mišićnog preparata A. suum (kontrakcije su neosetljive na mekamilamin). Objašnjenje može biti veća osetljivost mišićne kontraktilne mašine na EFS u odnosu na nerve koji oslobađaju ACh. Kontrakcije A. suum dobijene sa EFS su ipak bile osetljive na 10 i 30μM GABA, što se može objasniti snažnom hiperpolarizacijom mišićnih i nervnih ćelija askarisa koju GABA izaziva...