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Parazitske nematode su značajni patogeni životinja i ljudi. Nažalost, lekovi koji se koriste u farmakoterapiji helmintoza nisu apsolutno efikasni i bezbedni protiv svih kombinacija vrsta parazita/domaćina. Postoji jasna i hitna potreba za novim efikasnim antinematodnim jedinjenjima. Ova potreba postaje sve važnija sa sve većim nivoom rezistencije parazita na postojeće lekove. Većina anthelminika deluje na neuromišićni sistem nematoda. Mi u projektu predviđamo da se dalje ispita neuromišićni sistem na modelima parazitske nematode Ascaris suum i slobodnoživuće nematode Caenorhabditis elegans. Fokusiraćemo se na ispitivanje kombinaciju novih (muskarinski i serotoninski receptori) i prethodno potvrđenih potencijalnih ciljnih mesta (jonotropni nACh i GABA receptori). Predlažemo da se odredi lokalizacija ovih receptora u somatskim mišićima A. suum metodom RNA-scope in situ hibridizacije. Farmakološki ćemo okarakterisati ove receptore koristeći kombinaciju studija pokretljivosti, testova faringijalnog pumpanja (C. elegans), testova kontrakcije nervnomišićnog preparata (A. suum) i elektrofiziološkim metodama “voltage“ i „current clamp“ sa dve elektrode (A. suum). Koristeći ove tehnike, ispitivaćemo sledeće: 1. Efikasnost kombinacija postojećih GABA-ergičnih i holinergičkih lekova u cilju poboljšanja efikasnosti; 2. Potencijalno nove mete za anthelmintike (muskarinski ACh receptor i serotonergički MOD 1 receptor); 3) Prenamenu postojećih lekova koji ispoljavaju potencijalna anthelmintička svojstva; 4) Antinematodna svojstva terpenoidnih aktivnih sastojaka (AI) biljnih esencijalnih ulja i potencijal kombinacija AI i postojećih lekova. Očekujemo da će naši rezultati pružiti precizniju karakterizaciju postojećih (holinergičkih i GABA-ergičnih) i potencijalnih (muskarinskih i seretonergičkih) receptora za antiparazitske lekove. Utvrdićemo da li neki stariji lekovi imaju potencijal da se ponovo koriste kao anthelmintici, samostalno ili u kombinacijama.

The control of parasitic nematode infections relies mostly on anthelmintics. The potential pharmacotherapeutic application of phytochemicals, in order to overcome parasite resistance and enhance the effect of existing drugs, is becoming increasingly important. The antinematodal effects of carveol was tested on the free-living nematode Caenorhabditis elegans and the neuromuscular preparation of the parasitic nematode Ascaris suum. Carveol caused spastic and geraniol atonic paralysis in C. elegans. In A. suum carveol potentiated contractions induced by acetylcholine (ACh) and this effect was confirmed with two-electrode voltage-clamp electrophysiology on the A. suum nicotinic acetylcholine receptor expressed in Xenopus oocytes. However, potentiating effect of carveol on ACh-induced contractions was partially sensitive to atropine, indicates a dominant nicotine effect but also the involvement of some muscarinic structures. The effects of carveol on the neuromuscular system of mammals are also specific. In micromolar concentrations, carveol acts as a non-competitive ACh antagonist on ileum contractions. Unlike atropine, it does not change the EC50 of ACh but reduces the amplitude of contractions. Carveol caused an increase in Electrical Field Stimulation-evoked contractions of the isolated rat diaphragm, but at higher concentrations it caused an inhibition. Also, carveol neutralized the mecamylamine-induced tetanic fade, indicating a possibly different pre-and post-synaptic action at the neuromuscular junction.

Rastući nivo rezistencije parazita na antiparazitske lekove, kao i rigorozni standardi za maksimalne nivoe rezidua lekova u jestivim životinjskim tkivima, predstavljaju značajan izazov za savremenu veterinarsku farmakoterapiju. Parazitske nematode uzrokuju značajan morbiditet i smrtnost životinja ali i ljudi širom sveta. Takođe, uzrok su velikih gubitaka u proizvodnji hrane. Kontrola infekcija ovim parazitima oslanja se na efikasnu primenu anthelmintika, ali učestala primena istih anthelmintičkih jedinjenja za masovnu hemoterapiju dovodi do razvoja rezistencije na lekove. Konstantno postoji potreba da se identifikuju nova jedinjenja za razvoj potencijalnih anthelmintika. Sa drige strane, neuromišićni sistem parazitskih nematoda se pokazao kao efikasna farmakološka meta za za ove lekove. Neki od najčešće korišćenih antiparazitskih lekova su agonisti nikotinskih acetilholinskih receptora (nAChRs) (imidazotiazoli i tetrahidropirimidini) ili aktivatori glutamat-zavisnih hloridnih kanala (GluCls) i GABA-receptora (makrociklični laktoni). Holinergički agonisti kao što su levamisol, pirantel i oksantel selektivno otvaraju ligand-zavisne acetilholinske jonske kanale eksprimirane u mišićima zida tela nematoda da izazovu spastičnu kontrakciju mišićnih ćelija što dovodi do paralize crva. Predlažemo u cilju poboljšanja postojeće farmakoterapije i razvoja novih lekova istraživanja u sledećim pravcima: 1. Istraživanje efikasnosti kombinacija postojećih GABAergičnih i holinergičkih lekova za povećanje efikasnosti; 2. Istraživanje potencijalnih novih meta za anthelmintike (muskarinski ACh receptor i serotonergički MOD 1 receptor); 3) Novu namenu postojećih lekova, namenjenim drugim indikacijama koji ispoljavaju potencijalna antihelmintička svojstva; 4) Antinematodna svojstva terpenoidnih aktivnih sastojaka (AI) biljnih esencijalnih ulja i potencijalne kombinacija AI i postojećih lekova.

Culex mosquitos are important infectious diseases vectors in temperate and tropical regions of the World, affecting nearly 350 million people in both developed and developing countries. Our approach was to “recycle” the well-established larvicide, and by studying the tablets, pellets and granules as floating or sinking formulations, we found a method to optimise the use of diflubenzuron against Culex pipiens mosquitoes in field conditions.A standard WHO procedure was used to test the larvicide efficacy. The combined efficacy of all floating formulations was 10.7% higher than sinking preparations (p-value =0.002) and that maximised throughout the experiment on days 14 and 21, (p-values 0.012 and 0.008, respectively). All floating formulations kept their efficacies above 70% until day 21 of the experiment, while the mortality of sinking designs dropped significantly after day 14. The lowest efficacy was observed when sinking granules were used and the highest when floating tablets were applied in the canals. Only the floating tablets showed no significant changes in efficacy from day 1 to 21, but then that efficacy drops sharp until day 35.Since the larvae spend most of their time on the surface of the slow-moving waters to breathe, and floating pellets and tablets are made of materials that can serve as food sources, we conclude that these formulations have a higher effi cacy, at least in conditions of non- or slow-moving waters. This study shows the importance of a systematic approach to reformulation of old, already proven and environmentally safe larvicides which can control the mosquito populations and their spreading of various pathogens.

U veterinarskoj medicini postoji potreba za individualnom terapijom i terapijom stada/krda/jata, kada se istovremeno tretira veći broj životinja. Sprovođenje individualne terapije uglavnom ne predstavlja veći problem, ali istovremena primena lekova kod velikog broja životinja zahteva posebnu opreznost, precizno doziranje i potrebu da životinje dobiju adekvatnu dozu leka. Ovo je posebno teško sprovesti kada postoji potreba za tretiranjem krda divljih životinja. Naša iskustva se odnose na primenu antiparazitskih lekova kod jelenske divljači (Cervus elaphus i Dama dama) u ograđenom i otvorenom lovištu i divljih svinja (Sus scrofa) u gaterima. U tretmanu smo koristili benzimidazole, triklabendazol i albendazol kod jelena i ivermektin kod divljih svinja. Lekove smo primenjivali nanete na kukuruz, u blisterima soli, odnosno umešane u prekrupu (ivermektin). Osnovni problemi na koje smo nailazili su bili: smanjena palatibilnost hrane na koju je nanešen lek, problem kontrole količine leka koju su životinje unosile (kontrola doziranja), osetljivost lekova na tehnološke procese u blistiranju soli kao i promene na samim solištima koje smo morali da obavimo da bi obezbedili postojanost leka. Naša iskustva ukazuju da je potrebno najmanje jedna do dve nedelje navikavanja životinja na nove uslove prihrane da bi se posle tog perioda mogla odrediti doza leka i dužina tretmana. U ovom radu detaljno su prikazane tehnike davanja antiparazitskih lekova divljim papkarima hranom i efikasnost ovakve terapije.

Culex mosquitos are important infectious diseases vectors in temperate and tropical regions of the World, affecting nearly 350 million people in both developed and developing countries. Our approach was to “recycle” the well-established larvicide, and by studying the tablets, pellets and granules as floating or sinking formulations, we found a method to optimise the use of diflubenzuron against Culex pipiens mosquitoes in field conditions.A standard WHO procedure was used to test the larvicide efficacy. The combined efficacy of all floating formulations was 10.7% higher than sinking preparations (p-value =0.002) and that maximised throughout the experiment on days 14 and 21, (p-values 0.012 and 0.008, respectively). All floating formulations kept their efficacies above 70% until day 21 of the experiment, while the mortality of sinking designs dropped significantly after day 14. The lowest efficacy was observed when sinking granules were used and the highest when floating tablets were applied in the canals. Only the floating tablets showed no significant changes in efficacy from day 1 to 21, but then that efficacy drops sharp until day 35.Since the larvae spend most of their time on the surface of the slow-moving waters to breathe, and floating pellets and tablets are made of materials that can serve as food sources, we conclude that these formulations have a higher effi cacy, at least in conditions of non- or slow-moving waters. This study shows the importance of a systematic approach to reformulation of old, already proven and environmentally safe larvicides which can control the mosquito populations and their spreading of various pathogens.

Resistance of parasitic nematodes to anthelmintic drugs is a growing problem in human and veterinary medicine. The molecular mechanisms by which nematodes become resistant are different, but certainly one of the possible processes involves changing the drug binding site on the specific receptor. The significance of changes in individual subtypes of nicotinic acetylcholine receptors (nAChRs) for the development of resistance has not been clarified in detail. This study investigates the interaction of antinematodal drugs, agonist of different types of nAChRs and carvacrol with gamma aminobutyric acid (GABA) on the contractions of parasitic nematode A. suum. In our study, GABA (3 μM) produced significant increase of contractile EC50 value for pyrantel, and nonsignificant for bephenium and morantel, from 8.44 to 28.11 nM, 0.62 to 0.96 µM, and 3.72 to 5.69 nM, respectively. On the other hand, the maximal contractile effect (Rmax) did not change in the presence of GABA. However, when A. summ muscle flaps were incubated with GABA 3 μM and carvacrol 100 μM, the EC50 value of pyrantel, bephenium, and morantel was increased significantly to 44.62 nM, 1.40 μM, and nonsignificantly to 7.94 nM, respectively. Furthermore, Rmax decreased by 70, 60, and 65%. Presented results indicate that the combined use of GABA receptor agonists and nicotinic receptor antagonists can effectively inhibit the neuromuscular system of nematodes, even when one of the nicotinic receptor subtypes is dysfunctional, due to the potential development of resistance.

Parascaris sp. is the only ascarid parasitic nematode in equids and one of the most threatening infectious organisms in horses. Only a limited number of compounds are available for treatment of horse helminthiasis, and Parascaris sp. worms have developed resistance to the three major anthelmintic families. In order to overcome the appearance of resistance, there is an urgent need for new therapeutic strategies. The active ingredients of herbal essential oils are potentially effective antiparasitic drugs. Carvacrol is one of the principal chemicals of essential oil from Origanum, Thymus, Coridothymus, Thymbra, Satureja and Lippia herbs. However, the antiparasitic mode of action of carvacrol is poorly understood. Here, the objective of the work was to characterize the activity of carvacrol on Parascaris sp. nicotinic acetylcholine receptor (nAChR) function both in vivo with the use of worm neuromuscular flap preparations and in vitro with two-electrode voltage-clamp electrophysiology on nAChRs expressed in Xenopus oocytes. We developed a neuromuscular contraction assay for Parascaris body flaps and obtained acetylcholine concentration-dependent contraction responses. Strikingly, we observed that 300 µM carvacrol fully and irreversibly abolished Parascaris sp. muscle contractions elicited by acetylcholine. Similarly, carvacrol antagonized acetylcholine-induced currents from both the nicotine-sensitive AChR and the morantel-sensitive AChR subtypes. Thus, we show for the first time that body muscle flap preparation is a tractable approach to investigating the pharmacology of Parascaris sp. neuromuscular system. Our results suggest an intriguing mode of action for carvacrol, being a potent antagonist of muscle nAChRs of Parascaris sp. worms, which may account for its antiparasitic potency.

Three pharmacological subtypes of cholinergic receptors have been distinguished in Ascaris suum using a muscle contraction assay and classical pharmacological techniques. The receptor subtypes are: a B-subtype (sensitive to bephenium); an L-subtype (sensitive to levamisole and pyrantel); and an N-subtype (sensitive to nicotine and methyridine). Oxantel is a cholinergic anthelmintic that was first introduced for the treatment of whipworm, Trichuris, infections in children. Here, we compare the subtype selectivity of oxantel with thenium and other cholinergic anthelmintics. We used the A. suum assay to derive pA2 values for the agonists: oxantel, thenium, bephenium, levamisole, pyrantel, nicotine and methyridine with the antagonists: paraherquamide, 2-desoxyparaherquamide and methyllycaconitine. pA2 values, rather than pKB values, were determined for all agonists when it was found that Schild slopes for some agonists were significantly less than 1.0. The pA2 of oxantel was 6.58 ^ 0.25 for paraherquamide; 5.39 ^ 0.28 for 2-desoxyparaherquamide; 7.01 ^ 0.19 for methyllycaconitine. Comparison of pA2 values using cluster analysis showed that oxantel was grouped with nicotine and methyridine, the N-subtype agonists. Thenium had pA2s of 7.84 ^ 0.41 for paraherquamide; 5.52 ^ 0.50 for 2-desoxyparaherquamide; 6.33 ^ 0.19 for methyllycaconitine. Cluster analysis placed thenium between the L-subtype agonists and the B-subtype agonist. The therapeutic significance of classification of cholinergic anthelmintics is discussed. Combination of oxantel and pyrantel would have therapeutic advantages, covering N- and L-subtypes, and so increasing spectrum of action and reducing the potential for development of resistance. Our results predict that oxantel may remain effective in some nematode isolates that have become levamisole- and pyrantel-resistant.