Biol. Pharm. Bull. 25(12) 1651—1654 (2002) 1651
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December 2002 Notes Biol. Pharm. Bull. 25(12) 1651—1654 (2002) 1651 Inhibitory Effect of Isoquinoline Alkaloids 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, 5 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), chelerythrine and berberine 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 alkaloid; 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 protopine 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). Coptisine, 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) Sanguinarine 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. papaverine 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. 6 6 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 (2862), 6- HL60 cells were obtained from the RIKEN Cell Bank. MS (1861) and sanguinarine (5863) (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 (4196 HL60 cells were washed and resuspended in the culture 38), allocryptopine (349670), dehydrocorydaline (2956 medium at 33104 cells/ml, and 196 ml of this cell suspension 8), berberine (393610), emetine (109629), papaverine was placed in each well of a 96-well flat-bottom plate. The (250623) and CPDD (21006284). 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- chelidonine, 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,