December 2002 Notes Biol. Pharm. Bull. 25(12) 1651—1654 (2002) 1651

Inhibitory Effect of on Movement of Second-Stage Larvae of Toxocara canis

a b a a b Tadaaki SATOU, Nobuaki AKAO, Rinako MATSUHASHI, Kazuo KOIKE, Koichiro FUJITA, and ,a Tamotsu NIKAIDO* a Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan: and b Section of Environmental Parasitology, Division of Public Health, Graduate School of Tokyo Medical and Dental University; 1–5–45 Yushima Bunkyo-ku, Tokyo 113–8519, Japan. Received June 4, 2002; accepted August 27, 2002

To find new anthelmintics against parasites living in host tissues, we used an in vitro assay to screen iso- quinoline alkaloids for nematocidal activity on the larva of dog roundworm, Toxocara canis. To evaluate the effi- cacy of anthelmintics in vitro, Tsuda et al. previously introduced the concept of Relative Mobility (RM) of Toxo- cara larvae. After improvement of the assay system using image data processing, we generated a new index, ؍ RM50, the concentration at which RM 50%. However, except for pyrantel, the RM50 of most existing an- thelmintics could not be calculated because of low activity. Of the isoquinoline alkaloids tested, emetine, san- guinarine, 6-methoxydihydrosanguinarine (6-MS), and showed strong nematocidal ac- tivities. However, these compounds were highly cytotoxic; thus, the prospect of their direct application is low. We

then tested the cytotoxicity (IC50) of other isoquinoline alkaloids in HL60 tissue-culture cells. We continued our search for new anthelmintics by examining in detail the relationship between RM50 and IC50. We determined that an ideal target compound would exhibit a low RM50/IC50 ratio. Allocryptopine, dehydrocorydaline and papaver- ine were identified as potentially effective anthelmintics. Key words nematocidal activity; isoquinoline ; Toxocara canis; larva migrans; anthelmintics

Because few anthelmintics are effective against nema- MATERIALS AND METHODS todes, the diseases caused by the migration of nematodes into human tissues are usually very difficult to cure. Many known Anthelmintics Thiabendazole, a toxocariasis remedy, agents are effective only against nematodes living in the gas- was bought from Tokyo Kasei. Diethylcarbamazine, iver- trointestinal tract. In certain important nematodiasis, infec- mectin and albendazole were obtained from Sigma. Pyrantel tious larvae migrate to human tissues such as the liver, lungs, pamoate, an ascaridiasis remedy, was obtained from Sigma, heart, eyes, brain, or muscles through blood vessels, causing and piperazine was purchased from Wako. Kainic acid, an the characteristic persistent hypereosinophilia, hypergamma- anthelmintic targeting intestinal tract parasites, was pur- globulinemia, hepatomegaly and pneumonitis. In very seri- chased from Wako, and santonin was purchased from ous but rare cases, the hosts are killed. The development of a Aldrich. remedy is thus a pressing need. To find new anthelmintics Cytotoxic Agents Two anticancer agents, cis-Platinum(II) against parasites living in host tissues, we used an in vitro diammine dichloride (CPDD), obtained from Sigma, and screening assay to define the nematocidal activity of plant etoposide, purchased from Wako, were used for comparison derivatives on larvae of the dog roundworm, Toxocara canis, of cytotoxicity in vitro. the most common pathogenic parasite of visceral larva mi- Isoquinoline Alkaloids 6-Methoxydihydrochelerythrine grans. To evaluate the efficacy of anthelmintics in vitro, (6-MC), 6-methoxydihydrosanguinarine (6-MS), oxysan- Tsuda et al. introduced the concept of Relative Mobility guinarine, allocryptopine and were isolated from (RM) of T. canis larvae.1,2) Tsuda’s group has screened many the upper part of the Macleaya cordata plant, and their struc- plant extracts and evaluated their activity by dye exclusion tures were identified according to published data.3—6) d-Che- assay. Compounds showing strong nematocidal activity and lidonine was obtained from the upper part of the Chelido- the unusual effect of larval “bursting” have been found. nium majus plant, and its structure was confirmed by com- However, in vivo experiments have not been successful. parison with data in the literature.7) The air-dried tubers of In the present study, we improved the assay method devel- Corydalis turtschaninovii (Corydalis Tuber)8) were bought oped by Tsuda et al. and applied it to an in vivo experiment from Uchida (Japan). , dehydrocorydaline, d-cory- that examined nematocidal activity. To improve the result’s daline, l-stylopine, l-tetrahydrocolumbamine, and dl-tetrahy- objectivity, larval activity was recorded by a digital camera dropalmatine were obtained from the tuber of this plant, over a fixed time period, enabling continuous observation. which is also known as the Chinese crude drug “Corydalis After improvement of this assay system by image data pro- Tuber”.9—12) and chelerythrine were derived cessing, we estimated a new index, RM50, the concentration from 6-methoxydihydrosanguinarine and 6-methoxydihy- at which the RM equals 50% of the control. This system al- drochelerythrine, respectively, by hydrolysis.3,4) The structure lowed us to compare larval activity over a wider concentra- and purity of the isolated isoquinoline alkaloids were deter- tion range. In the present report, we describe the results from mined by TLC, melting point, UV, IR, optical rotation, and a study of isoquinoline alkaloids. The activities of these com- 1H-NMR. In addition to the compounds isolated from plant pounds were examined in detail and compared with existing materials, the isoquinoline alkaloids berberine, emetine, and anthelmintics. were examined. These three compounds were ob-

* To whom correspondence should be addressed. e-mail: [email protected] © 2002 Pharmaceutical Society of Japan 1652 Vol. 25, No. 12 tained from Sigma. (IC50) was calculated. The IC50 was derived from the mean of Second stage larvae of T. canis were collected by a previ- three replicates. ously described method and kept in Eagle’s MEM1 medium (Nissui Pharmaceutical) at 37 °C.1) The medium was changed RESULTS AND DISCUSSION weekly. Under these conditions, larvae of appropriate age are readily available for the assays and the culture can be main- We examined the nematocidal activity and cytotoxicity of tained for roughly 2 years. several isoquinoline alkaloids and compared them with exist- Nematocidal activity was determined according to a previ- ing anthelmintics. RM values of the compounds were com- ously described method.1) The test compounds were dis- pared at 24 h at the highest concentration practical. We also solved in a small volume of dimethyl sulfoxide (DMSO), evaluated nematocidal effects by comparing the RM50 values. which was diluted in 0.75% saline to 2% (v/v). For one assay, Each isoquinoline alkaloid or anthelmintic was tested, and 20 second-stage T. canis larvae were incubated with a test so- activity was examined 24 h after addition of the compounds. lution in a 96-well flat-bottom plate at 37 °C, and the behav- We were unable to calculate the RM50 values at any time ior of the larvae was observed under a microscope at 24 h point for any of the anthelmintics except pyrantel pamoate. Ϯ Ϯ after the start of incubation. All assays were repeated four However, pyrantel pamoate (means of RM50 S.D.: 46 3 times. Nematocidal activity was evaluated in terms of RM: a mmol/l) is effective only against an intestinal tract parasite lower RM indicates stronger nematocidal activity, and when and not against migrating larvae. These results demonstrate all the larvae die, the RM is zero. RM50 was calculated using the difficulty in identifying an effective remedy for larva mi- a calibration curve. This enabled us to easily compare the po- grans. Of the isoquinoline alkaloids, particularly strong ne- tency of the compounds over a wide concentration range. matocidal activity was observed for chelerythrine (28Ϯ2), 6- HL60 cells were obtained from the RIKEN Cell Bank. MS (18Ϯ1) and sanguinarine (58Ϯ3) (Fig. 1). However, These cells were maintained in RPMI 1640 medium (GIBCO these compounds exhibit high cytotoxicity activity, and their RBL) containing 10% fetal bovine serum (Sanko Junyaku) effects in vivo are uncertain. New anthelmintics cannot be supplemented with L-glutamine, 100 units/ml penicillin selected based on their nematocidal activity alone. Next, (Meiji Seika), and 100 mg/ml streptomycin (Meiji Seika). moderate nematocidal activity was observed in 6-MC (419Ϯ HL60 cells were washed and resuspended in the culture 38), allocryptopine (349Ϯ70), dehydrocorydaline (295Ϯ medium at 3ϫ104 cells/ml, and 196 ml of this cell suspension 8), berberine (393Ϯ10), emetine (109Ϯ29), papaverine was placed in each well of a 96-well flat-bottom plate. The (250Ϯ23) and CPDD (2100Ϯ284). On the other hand, no ne- cells were incubated in 5% CO2/air for 24 h at 37 °C. Test matocidal activity was observed in isoquinoline alkaloids: d- compounds were diluted in EtOH–H2O (1 : 1) to final con- , oxysanguinarine, protopine, coptisine, d-cory- centrations of 0.01—100 mmol/l, depending on the com- daline, l-stylopine, l-tetrahydrocolumbamine and dl-tetrahy- pound’s solubility. After incubation, 4 ml of these dilutions dropalmatine, anthelmintics: thiabendazole, albendazole, were added to the test wells, and 4 ml of EtOH–H2O (1 : 1) ivermectin, diethylcarbamazine, piperazine, kainic acid and was added into the control wells. The cells were incubated santonin, and cytotoxycity agent: etoposide in the judgment for 72 h in the presence of each agent, and cell growth was standard of RM50. evaluated using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5- In an attempt to identify compounds with low toxicity in 13) diphenyltetazolium bromide (MTT) reduction assay. vivo, we determined IC50 values using tissue-culture cells Briefly, after termination of the cell culture, 10 ml of 5 mg/ml HL60 in the above mentioned nematocidal compounds (Fig. MTT in phosphate-buffered saline (PBS) was added to each 2). Strong cytotoxicity activity was not observed in allocryp- Ϯ Ϯ well, and the plate was incubated in 5% CO2/air for 4 h at topine (means of IC50 S.D.: 48 9 mmol/l), dehydrocoryda- 37 °C. The plate was read on a microplate reader at 550 nm. line (19Ϯ1), or papaverine (14Ϯ0) as in pyrantel pamoate. A dose–response curve was plotted for compounds that These compounds will become lead chemical compounds in showed more than 50% cell growth inhibition at a sample the search for a remedy. There was similarly high cytotoxic- concentration, and the concentration causing 50% inhibition ity with CPDD (0.2Ϯ0.03) in chelerythrine (0.2Ϯ0.02), 6-

Ϯ Fig. 1. Means of RM50 (mmol/l) S.D. after 24 h

The chemical compounds are listed vertically, and the quadrature axis is a logarithmic display of RM50 in the second stage larvae of T. canis. December 2002 1653

Ϯ Fig. 2. Means of IC50 (mmol/l) S.D. after 24 h

The vertical line is shown chemical compounds are listed vertically, and the quadrature axis is a logarithmic display of IC50 in HL60 cultured cells.

Fig. 3. Means of RM50/IC50 after 24 h

The vertical line is shown chemical compounds are listed vertically, and the quadrature axis is a logarithmic display of RM50/IC50.

MC (0.2Ϯ0), 6-MS (0.3Ϯ0.02), sanguinarine (0.5Ϯ0), and ability etc. by adding to the anti-nematode activity. An in emetine (0.03Ϯ0.007). vivo experiment is progressing at present. There is no drug for larva migrans by T. canis etc. Alben- dazole, though it demonstrates some therapeutic effect,14) has Acknowledgements We thank Prof. Yutaka Sashida, Dr. weak action against the larva mentioned above. Therefore, Yoshihiro Mimaki, and Mr. Akihito Yokosuka of Tokyo Uni- pyrantel, with strong action on the larva, was used as a posi- versity of Pharmacy and Life Science for conducting the cy- tive control. We examined the relationship between RM50 and totoxicity assays. IC50 in greater detail in an attempt to improve the identifica- tion of new anthelmintics (Fig. 3). In this theory, a compound REFERENCES with an IC50 value that is much higher than its RM50 value would exhibit nematocidal activity without cytotoxicity. In 1) Kiuchi F., Miyashita N., Tsuda Y., Kondo K., Yoshimura H., Chem. screening isoquinoline alkaloids using Strongyloides ratti Pharm. Bull., 35, 2880—2886 (1987). and S. venezuelensis, this approach has been reported.15) 2) Akao N., Fukunaga M., Kondo K., Tsuda Y., Jpn. J. Parasitol., 41, Pyrantel pamoate (means of RM /IC : 0.5) showed the low- 519—526 (1992). 50 50 3) Hanaoka M., Motonishi T., Mukai C., J. Chem. Soc. Perkin Trans. 1, est RM50/IC50 ratios. Next, allocryptopine (7), dehydrocory- 12, 2253—2256 (1986). daline (16), and papaverine (17) showed low RM50/IC50 ra- 4) Itokawa H., Ikuta A., Tsutsui N., Ishiguro I., Phytochemistry, 17, tios. So allocryptopine, dehydrocorydaline, and papaverine 839—840 (1978). will be the candidate anthelmintics. The structural correla- 5) Pandey V. B., Ray A. B., Dasgupta B., Phytochemistry, 18, 695—696 tion could not be recognized. On the other hand, the ratios of (1979). 6) Zhang G. L., Rucker G., Breitmaier E., Mayer R., Phytochemistry, 40, 6-MC (1881), emetine (4127), and CPDD (8618) were very 1813—1816 (1995). high throughout this experiment. 7) Blanco O., Castedo L., Cortes D., Villaverde C., Phytochemistry, 30, 2071—2074 (1991). The RM50/IC50 ratios offered better differentiation than RM only. The present method should be able to help de- 8) Matsuda H., Tokuoka K., Wu J., Shiomoto H., Kubo M., Biol. Pharm. 50 Bull., 20, 431—434 (1997). velop a new drug. Since the its permeability is bad, pyrantel 9) Hanaoka M., Yoshida S., Mukai C., Chem. Pharm. Bull., 37, 3264— pamoate is effective against the intestinal parasite only. In 3267 (1989). vivo experiments are necessary in order to investigate perme- 10) Imaseki I., Taguchi H., Yakugaku Zasshi, 82, 1214—1219 (1961). 1654 Vol. 25, No. 12

11) Jewers K., Manchanda A. H., J. Chem. Soc. Perkin Trans. 2, 1972, Theyer I., Auer H., J. Ocular Pharmacol. Therapeutics, 17, 287—294 1393—1396 (1972). (2001). 12) Taguchi H., Imaseki I., Yakugaku Zasshi, 83, 578—581 (1963). 15) Satou T., Koga M., Matsuhashi R., Koike K., Tada I., Nikaido T., Vet- 13) Sargent J. M., Taylor C. G., Br. J. Cancer, 60, 206—210 (1989). erinary Parasitology, 104, 131—138 (2002). 14) Barisani A. T., Maca S. M., Hauff W., Kaminski S. L., Domanovits H.,