Assay of nematocidal activity of isoquinoline alkaloids using third-stage larvae of Strongyloides ratti and S. venezuelensis

Veterinary Parasitology 104 (2002) 131–138

Assay of nematocidal activity of isoquinoline alkaloids using third-stage larvae of Strongyloides ratti and S. venezuelensis Tadaaki Satou a , Masataka Koga b , Rinako Matsuhashi a , Kazuo Koike a , Isao Tada b , Tamotsu Nikaido a,∗ a

Department of Pharmacognosy, School of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan b Faculty of Medicine, Department of Parasitology, Kyushu University, Fukuoka 8128582, Japan

Received 27 June 2001; received in revised form 10 October 2001; accepted 11 October 2001

Abstract We examined the effects of isoquinoline alkaloids in vitro in an effort to identify a treatment for Strongyloides stercoralis larva migrans in humans. Infective third-stage larvae of S. ratti and S. venezuelensis were used as model nematodes for S. stercoralis. Nematocidal activity was evaluated by the 50% paralysis concentration (PC50 ). Most of the tested isoquinoline alkaloids had activity for S. ratti and S. venezuelensis. We then evaluated in vitro cytotoxicity, which was the 50% inhibition concentration (IC50 ) of the compounds using HL60 tissue-culture cells. Three of the compounds (protopine, d-corydaline, and l-stylopine) which exhibited strong nematocidal activity, showed little cytotoxicity. In addition, we examined the relationship between nematocidal activity and cytotoxicity using the PC50 /IC50 ratio. A ratio equivalent to or lower than that calculated for the currently prescribed strongyloidosis treatments, ivermectin, albendazole and thiabendazole, was observed for allocryptopine, protopine, dehydrocorydaline, d-corydaline, l-stylopine, and papaverine. In contrast, the PC50 /IC50 ratios for protopine, d-corydaline, and l-stylopine were substantially more favorable. Therefore, protopine, d-corydaline, and l-stylopine were identified as potential effective treatments for strongyloidosis. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Strongyloides ratti; S. venezuelensis; Isoquinoline alkaloid; Nematocidal activity; HL60 cells; Cytotoxicity

∗ Corresponding author. Tel.: +81-47-472-1391; fax: +81-47-472-1404. E-mail address: [email protected] (T. Nikaido).

0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 6 1 9 - 7

132

T. Satou et al. / Veterinary Parasitology 104 (2002) 131–138

1. Introduction Strongyloides stercoralis is a little studied parasite that infects no fewer than 100 million people worldwide, generally in regions between latitudes 35◦ N and 30◦ S. The distribution is limited primarily to warm moist areas because such climates are favorable to the survival of the larval stages (Maruyama et al., 1996; Nolan et al., 1999). Thiabendazole, albendazole, and ivermectin are the anthelmintics primarily used to treat this infection in humans. Cure rates range from 83% for ivermectin to 45% for albendazole. Ivermectin is presently the best drug available against S. stercoralis both in the clinical and primary health care setting. But, many agents are effective only against worms living in the gastrointestinal tract. Indeed very few anthelmintics are effective against nematodes living in the tissues. Moreover, Strongyloides hyperinfection is often diagnosed too late and sometimes found at necropsy (Tanaka et al., 1996; Gann et al., 1994). To find new anthelmintics against parasites living in host tissues, we used an in vitro screening test to detect nematocidal activity of compounds from plant materials on the larvae of S. ratti and S. venezuelensis. 2. Materials and methods 2.1. Test compounds Anthelmintics. Ivermectin and albendazole were purchased from Sigma (St. Louis, MO), thiabendazole from Tokyo Kasei, and diethylcarbamazine and pyrantel also from Sigma. Piperazine and the intestinal parasite anthelmintic, kainic acid, were purchased from Wako. Santonin was purchased from Aldrich (Milwaukee, WI, USA). Cytotoxic agents. Two anticancer agents, cis-platinum (II) diammine dichloride (CPDD), obtained from Sigma, and etoposide, purchased from Wako, were used for comparison of cytotoxicity in vitro. Isoquinoline alkaloids. 6-Methoxydihydrochelerythrine, 6-methoxydihydrosanguinarine, oxysanguinarine, allocryptopine, and protopine were isolated from the upper part of the Macleaya cordata plant, and their structures were identified according to published data (Hanaoka et al., 1986; Itokawa et al., 1978; Pandey et al., 1979; Zhang et al., 1995). d-chelidonine was obtained from the upper part of the Chelidonium majus plant, and its structure was confirmed by comparison with data in the literature (Blanco et al., 1991). The air-dried tubers of Corydalis turtschaninovii (Corydalis Tuber) (Matsuda et al., 1997) were bought from Uchida (Japan). Coptisine, dehydrocorydaline, d-corydaline, l-stylopine, l-tetrahydrocolumbamine, and dl-tetrahydropalmatine were obtained from the tuber of this plant, which is also known as the Chinese crude drug “Corydalis Tuber” (Hanaoka et al., 1989; Imaseki and Taguchi, 1961; Jewers and Manchanda, 1972; Taguchi and Imaseki, 1963). Sanguinarine and chelerythrine were derived from 6-methoxydihydrosanguinarine and 6-methoxydihydrochelerythrine, respectively, by hydrolysis (Hanaoka et al., 1986; Itokawa et al., 1978). Structure and purity of the isolated isoquinoline alkaloids were determined using TLC, melting point, UV, IR, optical rotation, and 1 H NMR. In addition to the compounds isolated from plant materials, the isoquinoline alkaloids berberine, emetine, and papaverine were examined. These three compounds were obtained from Sigma.

T. Satou et al. / Veterinary Parasitology 104 (2002) 131–138

133

2.2. Parasites The S. ratti L3 used in the present study were the TMDU strain (from Tokyo Medical and Dental University, Japan), maintained in the laboratory of Kyushu University for about 20 years by serial passage through rats. The S. venezuelensis L3 were the Naha Okinawa Japan strain, from University of Ryukyus, Japan. S. ratti and S. venezuelensis from the feces of infected rats were cultured for four days by filter-paper culture (No. 2, Advantec). The larvae were harvested, and washed twice with deionized/distilled water, and then filtered through clothmesh (Nytrel TI Qualite 15, UGB, France) to remove inactive larvae. 2.3. Preparation of samples and incubation Each compound (Table 1) was weighed and dissolved in organic solvent or distilled water to the desired concentration. Filter paper half-disks (8 mm in diameter, thick circular paper disks cut in half, Adventec) were placed in a 96-well plate and impregnated with the test solution. The organic solvent or distilled water was allowed to completely evaporate to eliminate any potential adverse effects on larvae. We then added 50 ␮l of distilled water and 50 ␮l of larval suspension (20 larvae per well) to each well. Although the optimum survival temperature of Strongyloides infective larvae is 15 ◦ C, incubation was carried out at body temperature, 37 ◦ C, to simulate the in vivo environment. 2.4. Evaluation procedure It was determined that accurate assessment of larval movement would require application of an external stimulus. Investigations of suitable chemical and physical stimuli in a preliminary study revealed that optimum results could be obtained by observing larvae 30 s after addition of 100 ␮l hot water (50 ◦ C), after the liquid surface had stabilized. 2.5. Microscopic evaluation An inverted microscope (CK2, Olympus) was used for observation and a digital camera capable of filming for 10 s intervals (QV-8000SX, Casio) was used to record the microscope image. We took one still image 30 s after the addition of hot water, another image 10 s later, and then recorded video images for the following 10 s. Motility was determined as a ratio of number of moving larvae to total larvae (control = 100%). Image processing software (Adobe Photoshop 5.0) was used for counting larvae on the still images. We determined the velocity, a ratio of twist frequency at a fixed position, (control = 100%) using animation replay software (QuickTime 3, Apple Computer). Each experimental metric was normalized to the control. Larval condition was expressed as follows: viability(%) = motility(%) × velocity(%)/100. A dose-response curve was plotted for the fractions and compounds that showed more than 50% viability at a sample concentration, and the 50% paralysis concentration (PC50 ) was calculated. The PC50 was derived from the mean value of four replications.

134

T. Satou et al. / Veterinary Parasitology 104 (2002) 131–138

Table 1 Responses to isoquinoline alkaloids, anthelmintics and anticancer drugs: effects on S. ratti and S. venezuelensis third-stage larvae (PC50 -S.r. or -S.v.) and cytotoxicity (IC50 -HL60) to HL60 cells Compounds

PC50 -S.r. (␮M)a

PC50 -S.v. (␮M)a

IC50 -HL60 (␮M)b

The benzophenanthridine alkaloids Chelerythrine d-Chelidonine 6-Methoxydihydrochelerythrine 6-Methoxydihydrosanguinarine Oxysanguinarine Sanguinarine

220 11 110 290 >500 280

72 200 61 77 >500 60

0.24 1.1 0.22 0.28 >100 0.50

61 52

51 33

60 230 32

32 48 12

18 14 >500 >500

30 13 >500 32

The protopine alkaloids Allocryptopine Protopine The protoberberine alkaloids Berberine Coptisine Dehydrocorydaline The tetrahydroprotoberberine alkaloids d-Corydaline l-Stylopine l-Tetrahydrocolumbamine dl-Tetrahydropalmatine Other isoquinoline alkaloids Emetine Papaverine

34 54

27 3.0

Anthelmintics Albendazole Diethylcarbamazine Ivermectin Kainic acid Piperazine Pyrantel Santonin Thiabendazole

11 43 2.2 >500 >500 0.21 74 78

16 45 2.3 200 >500 0.25 170 190

Cytotoxicity agents CPDD Etoposide

110 >500

200 >500

48 >100 3.3 8.7 19 >100 >100 >100 37 0.026 14 0.25 >100 2.0 >100 >100 >100 >100 55 0.24 0.28

a Compounds were tested up to soluble concentration on nematocidal activities (PC -S.r. or -S.v.: 50% paralysis 50 concentration) measured after 24 h incubation. b Compounds were tested up to 100 ␮M on cytotoxicities (IC -HL60: 50% inhibitory concentration) measured 50 by MTT method after 72 h incubation.

2.6. Cell culture HL60 cells were obtained from RIKEN Cell Bank, Japan. These cells were maintained in RPMI 1640 medium (GIBCO RBL, USA) containing 10% fetal bovine serum (Sanko

T. Satou et al. / Veterinary Parasitology 104 (2002) 131–138

135

Junyaku) supplemented with l-glutamine, 100 units/ml penicillin (Meiji Seika), and 100 ␮g/ml streptomycin (Meiji Seika). 2.7. Cytotoxicity assay (Sargent and Taylor, 1989) The leukemia cells were washed and resuspended in the culture medium to 3 × 104 cells/ ml, and 196 ␮l of this cell suspension was placed in each well of a 96-well flat-bottom plate. The cells were incubated in 5% CO2 /air for 24 h at 37 ◦ C. The test compounds were diluted in EtOH–H2 O (1:1) to final concentrations of 0.01–100 ␮M, depending on the solubility of the compounds. After incubation, 4 ␮l of these dilutions were added to the test wells and 4 ␮l of EtOH–H2 O (1:1) was added into the control wells. The cells were incubated for 72 h in the presence of each agent, and cell growth was evaluated using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetazolium bromide (MTT) reduction assay. After termination of the cell culture, 10 ␮l of 5 mg/ml MTT in phosphate-buffered saline was added to each well and the plate was incubated in 5% CO2 /air for 4 h at 37 ◦ C. The plate was then centrifuged at 1500 × g for 5 min to precipitate the cells and MTT formazan. An aliquot of 150 ␮l of the supernatant was removed from each well, and 175 ␮l of DMSO was added to dissolve the MTT formazan crystals. The plate was mixed on a microshaker for 10 min and then read on a microplate reader at 550 nm. A dose-response curve was plotted for the fractions and compounds that showed more than 50% cell growth inhibition at a sample concentration, and the concentration causing 50% inhibition (IC50 ) was calculated. The IC50 was derived from the mean of three replicates.

3. Results and discussion We compared the PC50 value and larval condition for each anthelmintic (Table 1) after the 24 h incubation. The PC50 value of ivermectin, which was the most effective strongyloidosis treatment, was 2.2 ␮M in S. ratti and 2.3 ␮M in S. venezuelensis. Activity of albendazole was lower than that of ivermectin and thiabendazole was lower than albendazole. In addition, pyrantel exhibited the highest PC50 value of 0.21 ␮M in S. ratti and 0.25 mM in S. venezuelensis. In contrast, kainic acid and piperazine showed weak activities for both species. The nematocidal activity of isoquinoline alkaloids is shown in Table 1. In the case of S. ratti none of the compounds approached the strong activity of ivermectin. However, equivalent effects to albendazole and thiabendazole were observed for d-chelidonine, allocryptopine, protopine, berberine, dehydrocorydaline, d-corydaline, l-stylopine, emetine, and papaverine. For S. venezuelensis, strong activity was observed only with papaverine. Otherwise. d-chelidonine, oxysanguinarine and l-tetrahydrocolumbamine exhibited weak effects. Alocryptopine, d-chelidonine, protopine, dehydrocorydaline, berberine, d-corydaline, l-stylopine, emetine, and papaverine were the only isoquinoline alkaloids effective against both larval species. Because so many potential treatments were identified among the isoquinoline alkaloids, it was necessary to further eliminate all but the strongest prospects. Basing the evaluation of efficacy only on nematocidal activity was difficult because of the wide range in PC50 values.

136

T. Satou et al. / Veterinary Parasitology 104 (2002) 131–138

To further assess the applicability of the selected isoquinoline alkaloids, we evaluated their in vitro cytotoxicity using HL60 cells (Table 1). The cytotoxic agents CPDD and etoposide, with values of 0.24 and 0.28 ␮M, respectively, were examined for comparison. Of the anthelmintics, kainic acid, santonin, pyrantel, piperazine, and diethylcarbamazine. Showed no cytotoxic effects at concentrations up to 100 ␮M. In contrast, cytotoxicity from albendazole was equivalent to that from CPDD and etoposide. Likewise, high Table 2 PC50 /IC50 ratio of the tested compounds and comparison of PC50 values between S. ratti and S. venezuelensis Compounds The benzophenanthridine alkaloids Chelerythrine d-Chelidonine 6-Methoxydihydrochelerythrine 6-Methoxydihydrosanguinarine Oxysanguinarine Sanguinarine

PC50 -S.r./ IC50 -HL60a 927 10 472 1034 – 569

PC50 -S.v./ IC50 -HL60b

PC50 -S.r./ PC50 -S.v.c

305 178 274 276 – 121

3 0.1 2 4 – 5

The protopine alkaloids Allocryptopine Protopine

1 <1

1 <0.3

1 2

The protoberberine alkaloids Berberine Coptisine Dehydrocorydaline

18 27 2

10 6 1

2 5 3

<0.2 <0.1 – –

<0.3 <0.1 – 1

1 1 – >16 1 18

The tetrahydroprotoberberine alkaloids d-Corydaline l-Stylopine l-Tetrahydrocolumbamine dl-Tetrahydropalmatine Other isoquinoline alkaloids Emetine Papaverine

1299 4

1026 0.2

Anthelmintics Albendazole Diethylcarbamazine Ivermectin Kainic acid Piperazine Pyrantel Santonin Thiabendazole

45 <0.4 1 – <11 <0.002 <1 1

64 <0.5 1 2 <14 <0.003 <2 4

Cytotoxicity agents CPDD Etoposide

435 –

828 –

a

The PC50 -S.r./IC50 -HL60 ratios in S. ratti. The PC50 -S.v./IC50 -HL60 ratios in S. venezuelensis. c The ratios of PC in S. ratti and in S. venezuelensis. 50 b

1 1 1 >13 1 1 0.4 0.4 1 –

T. Satou et al. / Veterinary Parasitology 104 (2002) 131–138

137

cytotoxicity was observed after incubation with the isoquinoline alkaloids sanguinarine, chelerythrine, 6-methoxydihydrochelerythrine, and 6-methoxydihydrosanguinarine. Emetine was particularly cytotoxic, with an IC50 value of 0.026 ␮M. Cytotoxicity was not observed after treatment with oxysanguinarine, protopine, d-corydaline, l-stylopine, and l-tetrahydrocolumbaminene at concentrations up to 100 ␮M. Three compounds showed moderate nematocidal activity and no cytotoxicity (protopine, d-corydaline, and l-stylopine) in both species. In theory, an ideal compound would exhibit low cytotoxicity at high concentrations and high nematocidal activity at low concentrations. Thus, we examined the relationship between nematocidal activity and cytotoxicity in detail (Table 2) using the PC50 /IC50 ratio. The PC50 /IC50 ratios of thiabendazole and ivermectin were nearly identical, although a 100 fold difference was observed in PC50 . The PC50 /IC50 ratios for albendazole were 45 in S. ratti and 64 in S. venezuelensis. Substantially lower ratios were calculated for santonin, pyrantel, and diethylcarbamazine, which were similar to or less than those for thiabendazole and ivermectin. Pyrantel exhibited a very low ratio for both species (S. ratti <0.002, S. venezuelensis <0.003). The isoquinoline alkaloids allocryptopine, protopine, dehydrocorydaline, d-corydaline, l-stylopine, and papaverine also exhibited equivalent or lower ratios than the strongyloidosis remedies, ivermectin, albendazole and thiabendazole. The low ratios of protopine, d-corydaline, and l-stylopine were particularly notable (Table 2). The tested compounds almost had similar effects in both species, S. ratti and S. venezuelensis. However, four anthelmintics and the isoquinoline alkaloids (kainic acid, papaverine, d-chelidonine, and dl-tetrahydropalmatine) showed stark species-specific differences in efficacy. Kainic acid, papaverine and dl-tetrahydropalmatine were significantly more effective against S. venezuelensis than S. ratti. The opposite was observed for d-chelidonine. Despite many morphological and physiological similarities, these two species differ in their migration routes after infection and thus appear to differ in their susceptibility to anthelmintics and isoquinoline alkaloids. Our purpose is to find inexpensive drugs in plants extracts which are more effective than the present chemosynthetics (including antimicrobial drugs) available to treat S. stercoralis in humans. Toward this purpose, our data suggest the need for further in vivo examination of protopine, d-corydaline, and l-stylopine (Fig. 1).

Fig. 1. Chemical structures of protopine (the protopine alkaloid), d-corydaline and l-stylopine (the tetrahydroprotoberberine alkaloids).

138

T. Satou et al. / Veterinary Parasitology 104 (2002) 131–138

Acknowledgements We thank Prof. Dr. Yutaka Sashida, Dr. Yoshihiro Mimaki, and Mr. Akihito Yokosuka of Tokyo University of Pharmacy and Life Science for conducting the cytotoxicity assay. We also thank Prof. Y. Sato, Department of Parasitology, School of Medicine, University of the Ryukyus and Dr. T. Tegoshi, Department of Medical Zoology, Kyoto Prefectural College of Medicine, for supplying the original Strongyloides venezuelensis strain. References Blanco, O., Castedo, L., Cortes, D., Villaverde, C., 1991. Alkaloids from Sarcocapnos-saetabensis. Phytochemistry 30, 2071–2074. Gann, P.H., Neva, F.A., Gam, A.A., 1994. A randomized trial of single- and two-dose ivermectin versus thiabendazole for treatment of strongyloidiasis. J. Infect. Dis. 169, 1076–1079. Hanaoka, M., Motonishi, T., Mukai, C., 1986. Chemical transformation of protoberberines. Part 9. A biomimetic synthesis of oxychelerythrine dihydrochelerythrine and chelerythrine from berberine. J. Chem. Soc., Perkin Trans. 1 (12), 2253–2256. Hanaoka, M., Yoshida, S., Mukai, C., 1989. Chemical transformation of protoberberines. XV. A novel and efficient method for the introduction of alkyl groups on C-13 position in the protoberberine skeleton. Chem. Pharm. Bull. 37, 3264–3267. Imaseki, I., Taguchi, H., 1961. Studies on the components of Corydalis spp. I. Alkaloids of Chinese Corydalis. On the new bases corydalmine and dehydrocorydalmine. Yakugaku Zasshi 82, 1214–1219. Itokawa, H., Ikuta, A., Tsutsui, N., Ishiguro, I., 1978. Alkaloids and a sterol from Chelidonium japonicum. Phytochemistry 17, 839–840. Jewers, K., Manchanda, A.H., 1972. The proton magnetic resonance spectra of protoberberinium salts. J. Chem. Soc., Perkin Trans. 2, 1393–1396. Maruyama, H., Noda, S., Nawa, Y., 1996. Emerging problems of parasitic diseases in southern Kyushu, Japan. Jpn. J. Parasitol. 45, 192–200. Matsuda, H., Tokuoka, K., Wu, J., Shiomoto, H., Kubo, M., 1997. Inhibitory effects of dehydrocorydaline isolated from corydalis tuber against type I–IV allergic models. Biol. Pharm. Bull. 20, 431–434. Nolan, T.J., Bhopale, V.M., Schad, G.A., 1999. Hyperinfective strongyloidiasis: Strongyloides stercoralis undergoes an autoinfective burst in neonatal gerbils. J. Parasitol. 85, 286–289. Pandey, V.B., Ray, A.B., Dasgupta, B., 1979. Minor alkaloids of Fumaria indica seeds. Phytochemistry 18, 695– 696. Sargent, J.M., Taylor, C.G., 1989. Appraisal of the MTT assay as a rapid test of chemosensitivity in acute myeloid leukemia. Br. J. Cancer 60, 206–210. Taguchi, H., Imaseki, I., 1963. Studies on the components of Corydalis spp. II. Alkaloids of Crydalis ambigua Cham. et Schlecht. var. amurensis Maxim. (1). On tertiary alkaloids. Yakugaku Zasshi 83, 578–581. Tanaka, S., Okumura, Y., Maruyama, H., Ishikawa, N., Nawa, Y., 1996. A case of overwhelming strongyloidiasis cured by repeated administration of ivermectin. Jpn. J. Parasitol. 45, 152–156. Zhang, G.L., Rucker, G., Breitmaier, E., Mayer, R., 1995. Alkaloids from Hypecoum leptocarpum. Phytochemistry 40, 1813–1816.