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Home , Dithiazanine iodide, Levamisole, Morantel, Nitroscanate, Praziquantel, Pyrvinium

Rev. Bio!. Trop., 35(1): 73-76, 1987.

The response of adult and free-living stages of , in vitro, to

Sanjeev Kumar Department of Zoology, Institute of Advanced Studies, Meerut University, Meerut 250 005, U.P., India

(Received June 5, 1986)

Abstract: The in vitro response of adults (males and females) and free-living stages of Necator americanus one of the human , to a wide variety of 20 broad and narrow spectrum anthelmintics was tested. Almost al! the broad spectrum anthelmintics influenced males, females and free-living stages at different levels and showed good activity with ECs o values varying from about 0.0002 and 0.0007 mg/l for pamoate and tricofenol respectively to abou t 8.47 and 7.6 mg/l for tartrate and amoscanate re­ spectively. Certain drugs (emetine, and ) exerted their effect either on male or female or free-living stages at 10.0 mg/l leve!. It is concluded that either sex or life-cycle stage alone may not be an ef­ fective criteria for screening of anthelmintics which have been employed at large; and females of

(in particular those of N. americanus) should be taken into account for proposing EC. o as they were found to require relatively highest EC� o level in almost al! the instances studied presently.

Ouring a survey of parasites of teria of sereening of anthelrnintics has remained Meerut region, Necator americanus was found restrieted ehiefly to either free-living or adult to be one of the most prevalent human para­ stages of the nematode parasites. Tlús may sites. Various notable in vitro tests have been sometime lead to false results as different ant­ devised fo r sereening of anthelminties employing helrnintie concentrations (ECso) may be re­ parasitie adults (Cooperia punctata, Leland et quired to affect different sexes or life-cycle al., 1975; Nippostrongylus brasiliensis, Jenkins stages, which has been explored during present et al. , 1980, Jenkins and Carrington, 1982; investigations which were undertaken chiefly spiralis, J enkins and Carrington, depending upon this view. The in vitro response 1981), free-living juveniles (horse strongyle lar­ of male, female and third stage infective larvae vae, Levine, 1950; Obeliscoides cuniculi and (L:;) of N americanus to twenty widely used calcaratus, Tiner, 1958; eggs anthelmintics is now reported. to third stage � of N brasiliensis, Phillipson, 1966 and N brasilensis, Nematodirus dubius. Haemonchus contortus, Trichostrongylus colu­ MATERIALS ANO METHOOS briformis and Ostertagia ostertagi, Ibarra and J enkins, 1984 and the free-living nematode Motile N americanus adults were obtained (Simpkin and Coles, from the intestines during post-mortem exam­ 1981). In addition, literature (Steward, 1955; ination in local hospitals or from pathological Copp et al. , 1958; Sheffield etal. , 1959; Broome laboratories. The worms were washed thorough­ and Greenhalgh, 1961; Standen, 1963; Stone ly in physiological saline and the two sexes et al. , 1965; Katiyar and Sen, 1969; Coles and were separated. To obtain eggs the females were Me Neillie, 1977; Jenkins, 1982) reveals that no incubated in physiological saline at 370C. The study is available employing different sexes and eggs laid were washed at least thrice in distilled life-eycle stages altogether of a single nematode water and adjusted to a known density in water to investigate anthelmintie response. The eri- using the McMaster technique.

73 74 REVISTA DE BIOLOGIA TROPICAL

Male and female adults and eggs were ax­ arrested or slowed the development of the enized for 30 mino in warm (370C) salíne (0.15 larvae relative to the controls, were regarded as

M-NaCl) contaíning antibiotics (penicillín, 124 active. ECs o values were determíned by subject­ ¡Jg; streptomycin, 70 ¡Jg; mycostatin, 100 units ing the data obtaíned for the effects observed at per mI). 150 adult worms (males and females each serial concentration of drug where be­ separately) per 200 mI of culture medium in tween lO to 90% ofthe test worms were affected stoppered Erlenmeyer flasks or 5 ,000 eggs per relative to the controls, to línear regression mI of culture medium in stoppered glass analysis. contaíners on a roller drum, were incubated for 16 h (adult) or for 5 days (eggs) in predeter­ RESULTS mined (unpublíshed observatíons) optímal con­ ditions (5% CO2 , 10% O2 and 85% N2 ; 38 ± Results are summarised in Table 1. Out of 10C). The incubation medium consisted of 20 compounds studied 15 showed significant RPMI-1640 medium (Grand Island Biologícal activity (P < 0.01) relative to the control s at Company, NY,USA) supplemented withO.p225 concentrations below 10 mg/l against both mg per mI and antíbiotic mixture as adult (males and females) and juveníle stages. described aboye plus drug solution/ suspension. Out of 15 active compounds the most potent Water insoluble drugs were dissolved in a suitable were pyrantel pamoate and tricofenol pipera­ volatíle solvent (acetone or diethyl ether) and a zine which were fully active at the lowest con­ known volume was added to the container. centrations used and the least potent were After the solvent evaporated completely leaving amoscanate and morantel tartrate . , a thin film of drug deposit over the basal surface pamoate and thiabendazole were of each container, the culture medium was add­ moderately potent. Females and juveniles re­ ed. Culture medium controls were prepared and quired relatively maxímum and minimum drug treated identically with the exception that the concentrations respectively to show significant drugs were not included. Males, females and response. 100% adult worms survived in all eggs were incubated in the control culture media control incubation media and 96% worms were exactly as described before. - fully active after 16 h. AIl the 15 active drugs Each drug was tested initially at concentra­ exerted lethal effects on the adult worms at tions of 30, 20 and 10 mg/l whole compound. different concentrations. Females, however, Subsequently drugs showing activity at 10.0 showed maxímum resistance. After 16 h the mg/l were titrated in a retest using decrements worms that were alíve became greatly lethargic either 1: 2, 1: 3 or 1: 4 and so on in order to in all the 20 drugs. In ivermectin, pyrantel determine the concentrations required to affect pamoate and tricofenol piperazine live worms approximately 50% of the worm (ECs o ) accord­ were pale and highly coíled. Piperazine, diet­ ing to earlíer practices (Ibarra and Jenkins, hy1carbamazine and praziquant�l, however, 1984). The media were not changed during the exerted no lethal effects on either male or fe­ experiments. For each , observa­ maleo Praziquantel and suramin were slíghtly tions were carried out either macroscopically lethal to L3 and female respectively. Emetine (for adults) or under a binocular microscope at was a bit lethal to both female as well as L3 ' x 40 magnification (for eggs and larvae). The Both of them developed grossly abnormal ap­ number and physical condition of males, fe­ pearance. 99 - 100% control eggs attaíned full males, eggs and larvae present in each test and development into third ínfective larval stage control contaíners· were recorded on O h. The within 5 days. Compounds were regarded to adults were examined again after 16 h and the exert ovicidal effects where the eggs did not number of dead, paralysed and abnormal show development and hatching, larvicidal worms was recorded. Eggs were examined after effects when the larvae died during development 5 days and the number and condition of eggs and hatching or were inhibitory when develop­ and larvae present were recorded. Activity of ment of the larvae was either slowed down sig­ the compounds was assessed by comparing the nificantly or arrested completely. The drugs test worms with the controls. studied showed a variety of such activities. The drugs that killed, paralysed or made the Though thiabendazole, and worms greatly abnormal relative to controls or thiophanate showed no ovicidal activity even at that elícited ovicidal or larvicidal effects or concentrations as high as 30 mg/l, all the three KUMAR: Response of Necator americanus to anthelmintics 75

TABLE 1

Response of adult (males and females) and free-living stages of Necator americanus to 20 broad and na"ow spectrum anthelmintics in vitro. Approximate concentration mg/l required to affect 50% of the worms (EC,o)

Anthelmintic Life-cycle stage

Parasitic adult Free-living (L3) juvenile Male Female

Amoscanate 4.4 7.6* 3.8 Bephenium hydroxynaphthoate 0.47 ± 0.4 citrate 0.027 0.11 * G.02 Emetine ± ± Febantel 0.76 0.91 * 0.84 Ivermectin 0_004 0.005* 0.001 Levamisole HC1 0.021 0.074* 0.011 Morantel tartrate 5.81 8.47* 3.07 0.53 0.74* 0.43 0.024 0.14* 0.09 Piperazine hexahydrate Praziquantel + Pyrantel pamoate 0.0002 0.0005* 0.0003 Pyrvinium pamoate 0.006 0.009* 0.003 Stilbazium iodide 0.021 0.03* 0.017 Suramin + Thiabendazole 0.003 0.004* 0.0038 Thiophanate 0.84* 0.77 0.8 Tricofenol piperazine 0.008* 0.0007 0.006

* Highest (troe) EC,o for N. americanus species as a whole + Good activity, > 50 % test worms affected relative to controls at 10.0 mg/1 whole compound ± Weak activity, between 20 and 50 %worms affected relative to controls at 10.0 mg/l whole compound No activity, < 20 % test worms affected relative to controls at 10 mg/l whole compound

showed larvicidal activity. Ivennectin and adults and also provide a new criteria of screen. phenothiazine markedly inhibited the dev­ Even the two sexes of N americanus required elopment of larvae and their motility was great­ different drug concentrations to show- signifi­ ly reduced. At high concentrations ivennectin cant (P < 0.01) response. The relatively low and thiabendazole produced small blister like ECs o values of drugs for free-living stages if are patches on the larval body wall. In rest of the taken into account as criteria for selection of drugs larvae became lethargic. drug of choice for preparation of doses, it will result to insignificant effects and the probabili­ DISCUSSION ty of entire worm expulsion by intake of drug selected in this way by the host, is extremely Most of the available anthelmintic screen­ low. lt is therefore, considered that ECso values ing tests carried out in vitro are based on free­ obtained for females may be more reliably living life-cyc1e stages alone, Jenkins (1982) has, regarded to influence the species effectively. however, pointed out and discussed disadvan­ Since adult parasitic stages inhabit the host and tages and limitations of such screens and has particularly females are responsible for the stated that there is a possibility of getting spread of , it is more appropriate to "false positive or negative" results. Present screen and select the drugs taking their effect investigations provide experimental evidence on females as basic criteria. In few drugs, how­ that the ECso values obtained for free-living ever, EC so values were higher for males or L3 ' stages may not be reliably taken over to the In such cases the relatively highest ECso value 76 REVISTA DE BIOLOGIA TROPICAL among male, female or L3 is advisable. Jenkins, D, C. & T. S. Carrington. 1981. An in vitro The screen undertaken presentIy was highly screening test for compounds active against the specific as it was intended to detect the activity parenteral stages of Trichinella spiralis. Tropenmed­ izin und Parasitologie 32: 31-34. against N. americanus only. No ovicidal effect of thedrugs' was probably Jenkins, D. C. & T. S. Carrington. 1982. An in vitro due to the egg shell which serves as an effective screen for anthelmintics employing worms of barrier for the penetration of the drug. During Nippostrongylus brasiliensis in a defined medium. anti-trichostrongyle screens, levamisole and Veterinary 11: 223-230. morantel tartrate have been reported (Ibarra Jenkins, D. C., R. Armitage & T. S. Carrington, 1980. and Jenkins, 1984) to show no ovicidal effects A new primary screening test for anthelmintics even at concentrations as high as 50 mg/l but utilizing the parasite stages of Nippostrongylus both were markedly larvicidal and ivermectin to brasiliensis in vitro. Zeitschrift fUf Parasitenkunde markedly inhibit both development and motili­ 63: 261-269. ty of the larvae. Katiyar, J. C. & A. B. Sen. 1969. A new technique for rapid sereening of potential nematocidal com­ ACKNOWLEDGEMENTS pounds. Indian Journal of Parasitology 31: 132-1 34. Leland, S. E., G. R. Ridley, C. F. Slonka & G. L. The author is grateful to S. S. Lal and K. C. Zimmerman. 1975. Detection ofactivity for various Pandey, Meerut University, for the facilities. anthelmintics against in vitro produced Cooperia punctata, American Journal of Research in Veter­ RESUMEN inary Sciences 36: 449-456. Levine, N. D. 1950. The use of horse strongyle larvae Se probó la respuesta in vitro de Necator in screening compounds for anthelmintic aetivity. americanus a 20 antihelmínticos. Con valores Transactions of Illinois State Academy of Science de ECso desde 0.0002 hasta 0.0007 mg/L, los 43: 233-236. antihelmínticos de espectro amplio afectaron a Phillipson, R. F. 1966. Life-eycle and ehemotherapeu­ machos, hembras y estad íos libres. Ciertas dro­ tic studies on Nippostrongylus brasiliensis. Ph. D. gas (emetine, praziquantel y suramin) afectaron Thesis, University of London. selectivamente a los grupos mencionados al ni­ vel de 10.0 mg/L. Las hembras requirieron ma­ Sheffield, H. G., J. E. Meisenhelder & P. E. Thompson. 1959. The effects of referenee anthelmintics against yores niveles de tratamiento, lo que tiene im­ N. dubius and oxyurids in mice relative to screening portantes implicaciones en la aplicación de an­ procedures for new drugs, Journal of Parasitology tihelmínticos. 45: 653-6 58.

REFERENCES Simpkin, K, G. & G, C. Coles. 1981. The use of Ca enorhabditis elegans for anthelmintic screen­ ing, Journal of Chemical Techniques and Biotech­ Broome, AW. J. & N. Greenhalgh. 1961. A new anthel­ niques 31: 66-69. mintie with unusual properties. Nature 189: 59-60. Standen, O. D. 1963. Chemotherapy ol' helminthic Coles, G. C. & R, M. McMeillie. 1977. The response of , In "Experimental Chemotherapy" (R. nematodes in vivo and in vitro to some anthelmin­ 1. Sehnitzer & F. Hawking, eds.) pp. 701. Academ­ tics, J0l!rnal of Helminthology 51: 323-326. ic Press,

Copp, F. C., O. D., Standen, J. Scarnwell, D. A. Rawes Steward, J. S. 1955, Anthelmintic study: A double & R, B. Burrow, 1958. A new series of anthelmin­ enteronemacidal anthelmintic test covering a wide ties, Nature 181: 183, range of aetivities, Parasitology 45: 242-254,

Ibarra, O. F. & D. C. Jenkins, 1984. The relevance· of Stone, O. J" J. F. F. Mulins & C, C. Wells. In vitro in vitro anthelmintic sereening tests employing the studies on with thiabenda­ free-living stages of trichostrongylid nematodes. zole with observations on larval exsheathment. Journal of Helminthology 58: 107, Journal of Investigations on Dermatology 44: 256-258. Jenkins, D, C. 1982. In vitro screening tests for ant­ helmintics. In " Models in Parasitology" (D. Tiner, 1. D. 1958. A preliminar y in vitro test for ant­ G. Owen, ed,) pp. 173-186. Mc Millan Press, Lon­ helmintic activity. Experimental Parasitology 7: don, 292-305,