Journal of Helminthology (1979) 53, 31-33
The effect of Praziquantel on Schistosoma mansoni G. C. COLES Imperial Chemical Industries, Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire SK10 4TG ABSTRACT 3 x 10-6M praziquantel fails to completely paralyse miracidia and cercariae in a short time but they are not infective when maintained in the solution. 3 x 10-5M praziquantel prevents infected snails shedding cercariae but does not kill daughter2 sporocyts or developing cercariae. As the action of praziquantel on adult worms is not blocked by 10- M mecamylamine, pempidine or carbachol, but is reduced by calcium depletion, it is suggested that praziquantel may act by permitting calcium influx to muscle cells causing them to contract.
Praziquantel (2-cyclohexylcarbonyl - 1,3,4,6,7, llb-hexahydro - 2H - pyrazino [2-1-a] isoquinoline-4-one) is an interesting novel drug with activity against cestodes (Thomas and Gonnert, 1977) and schistosomes (Gonnert and Andrews, 1977). The drug causes paralysing contractions in tapeworms (Thomas and Andrews, 1977) and can immobilise cercariae and miracidia (Andrews, 1978) but its mechanism of action and its effects on all stages of schistosomes have not been reported. METHODS Schistosoma mansoni of Egyptian origin (Wellcome strain) was maintained in Brazilian Biomphalaria glabrata and Alderley Park strain white mice by standard laboratory procedures. Adult worms were collected from mice by perfusion with Tyrode's solution and maintained in this medium at 37°C for a maximum of 2 hours. Adult Fasciola hepatica were dissected from bile ducts of rats infected 12 weeks previously with 20 metacercariae each. RESULTS Miracidia Swimming behaviour changed on treatment with 3 X 10~6M praziquantel, the miracidia frequently altering course or swimming along a corkscrew-like path. The middle part of the miracidia contracted, giving the appearance of an unequal dumbell, with the greater mass anterior. Twenty young B. glabrata were individually exposed to 10 miracidia each in 3 x 10"6M praziquantel (0.5 ml volume). No snails became infected. Untreated miracidia from the same source were infective to control snails. Daughter Sporocysts
Infected snails, shedding cercariae, were maintained for 24 hours in 3 X 10~6 and 3 X 10"5M praziquantel before being returned to normal water. Snails were checked individually for shedding of cercariae before being dissected on day 7. A very few cercariae were shed by snails treated with 3 x 10-6M praziquantel and none from snails treated with 3 X 10 5M praziquantel. However, all snails were filled with daughter sporocysts and developing cercariae. Snails maintained for 24 hours in 3 x 10~6M praziquantel and dissected at 48 hours had numerous developing cercariae but relatively few with tails. 31
G. C. COLES
Cercariae At 10"6M praziquantel did not appear to rapidly affect cercariae, which swam normally, but at 3 X 10"6M cercariae immediately contracted strongly and ceased swimming. When maintained in the drug they gradually resumed movement but did not move sufficiently to swim from the bottom of the container. Tails were then shed, this occurring slightly faster at lower concentrations (Table 1), and the body of the cercariae crawled around. TABLE 1 The effect of praziquantel concentration on the rate of shedding of tails by cercariae of S. mansoni Percentage tails shed Fraziquantel concentration (M) 8
3 x 1010-5 5 3 x 1010-4
time in mins
10
30
60
90
5 3 0 0
29 5 1 5
65 52 44 42
91 91 87 91
Cercariae in Tyrode's solution or sucrose isotonic with Tyrode swam normally for considerable periods. If treated in these solutions with 3 x 10"6M praziquantel they sank to the bottom and appeared to shrivel up. The bodies and tails did not separate. After 40 minutes in sucrose and praziquantel only 1 % of cercariae showed slight occasional movement, whereas 92 % still showed slight movement in Tyrode and praziquantel. Mice exposed by the ring technique to cercariae in 3 x 10~8M praziquantel did not contain adult worms 7 weeks later. However, if cercariae were paralysed with 3 x 10 6M praziquantel for 2 minutes and then washed free of the drug they resumed normal swimming behaviour and were infective to mice. Adult Worms Adult worms placed in 3 x 10~6M praziquantel in Tyrode contracted strongly in about 30 seconds and failed to resume movement. By contrast adult F. hepatica were actively moving after 18 hours in 3 X 10~4M praziquantel in Hedon Fleig solution. Adult S. mansoni exposed to 3 x 10~6M praziquantel for 30, 60 or 120 seconds and washed in Tyrode partially resumed movement. Worms continued moving in 10~2M mecamylamine and 10"2M pempidine. Neither drug prevented the paralysis caused by praziquantel. 10^2M carbachol caused a relaxing paralysis of S. mansoni but 3 X 10"6M praziquantel caused the worms to contract. Atropine (10"2M) caused a partial contraction of worms but worms continued moving for over 2 minutes. Worms exposed to 10~2M atropine for 30 seconds then praziquantel (3 x 10"6M) ceased movement within 30 seconds. If dropped into distilled water adult worms rapidly paralyse. In 0.9 % sodium chloride worms remain moving over an extended period. An equivalent solution of potassium chloride more rapidly paralysed worms but a solution of calcium chloride (.078M) produced a fairly rapid paralysis. By contrast an equivalent solution of magnesium chloride caused worms to gradually relax and cease movement. Worms maintained in the magnesium saline for 5 minutes contracted when treated with praziquantel but gradually relaxed again. Worms maintained in a sodium, potassium chloride saline (NaCl .137M, KC1, 0.0027M) with or without 2mMEDTA for 20 minutes only contracted relatively weakly to praziquantel, those in EDTA responding less than those without EDTA. Three minutes after treatment with praziquantel worms were quite relaxed and some showed sluggish movements. 32
Effect of praziquantel on Schistosoma mansoni
DISCUSSION At similar concentrations the effect of praziquantel is more marked on adult S. mansoni than on larval stages. The daughter sporocysts are resistant to exposure of snails for 24 hours to 3 x 10 5M drug and the effects of short exposures of cercariae to the drug is fully reversible as judged by infectivity tests. The rapid contraction of cercariae in praziquantel with subsequent tachyphylactic type of response, as described for certain nematodes treated with levamisole, (Coles et ah, 1975), could possibly be due to an effect on the nervous system. However the shrivelling of cercariae treated with praziquantel in osmotically concentrated solutions suggests praziquantel affects the ionic or osmotic regulation of the cercariae. Experiments with adult worms confirm this latter interpretation. Four drugs known to affect the nervous system of helminths failed to prevent praziquantel induced contractions, suggesting the drug acts directly on the parasite musculature. Increased calcium ion concentrations caused schistosomes to contract and reduced calcium ion concentrations prevented or reduced the effect of praziquantel. A situation was reached in calcium free solutions where some movement still occurred after praziquantel treatment. Since praziquantel was not active on a calcium ionophore screen (unpublished observations) it is unlikely to be carrying calcium ions across the cell membrane. More probably praziquantel is opening pores in the membrane permitting a rapid influx of calcium ions, either directly or indirectly through an effect on sodium ions. The effect on ion movement and therefore motility is apparently reversible in cercariae (Andrews, 1978) and to a lesser extent in adult worms. A similar reversibility of paralysis in vitro has been observed with contracted tapeworms washed free of praziquantel (Thomas and Andrews, 1977). Detailed electrophysiological examinations of the effect of praziquantel on S. mansoni confirm that it directly or indirectly affects calcium ion movement in the worm (Bennett, 1977—personal communication). An effect on calcium ions could alter cell permeability and explain the leakage of glucose observed with praziquantel treated Hymenolepis diminuta (Thomas and Andrews, 1977). There seems no need to invoke a direct effect on carbohydrate metabolism to explain the drug's schistosomicidal and cestodicidal activity. Why the drug affects one group of trematodes, but not another (liver flukes) remains unclear. ACKNOWLEDGEMENT I wish to thank Dr. P. Andrews of Bayer AG for a gift of pure praziquantel. REFERENCES ANDREWS, P. (1978) Effect of praziquantel on the free living stages of Schistosoma mansoni. Zietschrift fur Parasitenkunde (in press). COLES, G. C , EAST, J. M. and JENKINS, S. W. (1975) The mechanism of action of the anthelmintic levamisole. General Pharmacology, 6, 309-313. GONNERT, R. and ANDREWS, P. (1977) Praziquantel, a new broad spectrum antischistosomal agent. Zeitschrift fur Parasitenkunde, 52, 129-150. PAX, R., BENNETT, J. L. and FETTERER, R. (1978) A benzodiazepine derivative and praziquantel: effects on musculature of Schistosoma mansoni and S.japonicum. Naunyn-Schmiederberg's Archives of Pharmacology, 304, 309-315. THOMAS, H. and ANDREWS, P. (1977) Praziquantel—a new cestodicide. Pesticide Science, 8, 556-560. THOMAS, H. and ANDREWS, P. (1977) The efficacy of praziquantel against cestodes in animals. Zeitschrift fur Parasitenkunde, 52, 117-127. Accepted1) February, 1978.
33
The effect of Praziquantel on Schistosoma mansoni G. C. COLES Imperial Chemical Industries, Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire SK10 4TG ABSTRACT 3 x 10-6M praziquantel fails to completely paralyse miracidia and cercariae in a short time but they are not infective when maintained in the solution. 3 x 10-5M praziquantel prevents infected snails shedding cercariae but does not kill daughter2 sporocyts or developing cercariae. As the action of praziquantel on adult worms is not blocked by 10- M mecamylamine, pempidine or carbachol, but is reduced by calcium depletion, it is suggested that praziquantel may act by permitting calcium influx to muscle cells causing them to contract.
Praziquantel (2-cyclohexylcarbonyl - 1,3,4,6,7, llb-hexahydro - 2H - pyrazino [2-1-a] isoquinoline-4-one) is an interesting novel drug with activity against cestodes (Thomas and Gonnert, 1977) and schistosomes (Gonnert and Andrews, 1977). The drug causes paralysing contractions in tapeworms (Thomas and Andrews, 1977) and can immobilise cercariae and miracidia (Andrews, 1978) but its mechanism of action and its effects on all stages of schistosomes have not been reported. METHODS Schistosoma mansoni of Egyptian origin (Wellcome strain) was maintained in Brazilian Biomphalaria glabrata and Alderley Park strain white mice by standard laboratory procedures. Adult worms were collected from mice by perfusion with Tyrode's solution and maintained in this medium at 37°C for a maximum of 2 hours. Adult Fasciola hepatica were dissected from bile ducts of rats infected 12 weeks previously with 20 metacercariae each. RESULTS Miracidia Swimming behaviour changed on treatment with 3 X 10~6M praziquantel, the miracidia frequently altering course or swimming along a corkscrew-like path. The middle part of the miracidia contracted, giving the appearance of an unequal dumbell, with the greater mass anterior. Twenty young B. glabrata were individually exposed to 10 miracidia each in 3 x 10"6M praziquantel (0.5 ml volume). No snails became infected. Untreated miracidia from the same source were infective to control snails. Daughter Sporocysts
Infected snails, shedding cercariae, were maintained for 24 hours in 3 X 10~6 and 3 X 10"5M praziquantel before being returned to normal water. Snails were checked individually for shedding of cercariae before being dissected on day 7. A very few cercariae were shed by snails treated with 3 x 10-6M praziquantel and none from snails treated with 3 X 10 5M praziquantel. However, all snails were filled with daughter sporocysts and developing cercariae. Snails maintained for 24 hours in 3 x 10~6M praziquantel and dissected at 48 hours had numerous developing cercariae but relatively few with tails. 31
G. C. COLES
Cercariae At 10"6M praziquantel did not appear to rapidly affect cercariae, which swam normally, but at 3 X 10"6M cercariae immediately contracted strongly and ceased swimming. When maintained in the drug they gradually resumed movement but did not move sufficiently to swim from the bottom of the container. Tails were then shed, this occurring slightly faster at lower concentrations (Table 1), and the body of the cercariae crawled around. TABLE 1 The effect of praziquantel concentration on the rate of shedding of tails by cercariae of S. mansoni Percentage tails shed Fraziquantel concentration (M) 8
3 x 1010-5 5 3 x 1010-4
time in mins
10
30
60
90
5 3 0 0
29 5 1 5
65 52 44 42
91 91 87 91
Cercariae in Tyrode's solution or sucrose isotonic with Tyrode swam normally for considerable periods. If treated in these solutions with 3 x 10"6M praziquantel they sank to the bottom and appeared to shrivel up. The bodies and tails did not separate. After 40 minutes in sucrose and praziquantel only 1 % of cercariae showed slight occasional movement, whereas 92 % still showed slight movement in Tyrode and praziquantel. Mice exposed by the ring technique to cercariae in 3 x 10~8M praziquantel did not contain adult worms 7 weeks later. However, if cercariae were paralysed with 3 x 10 6M praziquantel for 2 minutes and then washed free of the drug they resumed normal swimming behaviour and were infective to mice. Adult Worms Adult worms placed in 3 x 10~6M praziquantel in Tyrode contracted strongly in about 30 seconds and failed to resume movement. By contrast adult F. hepatica were actively moving after 18 hours in 3 X 10~4M praziquantel in Hedon Fleig solution. Adult S. mansoni exposed to 3 x 10~6M praziquantel for 30, 60 or 120 seconds and washed in Tyrode partially resumed movement. Worms continued moving in 10~2M mecamylamine and 10"2M pempidine. Neither drug prevented the paralysis caused by praziquantel. 10^2M carbachol caused a relaxing paralysis of S. mansoni but 3 X 10"6M praziquantel caused the worms to contract. Atropine (10"2M) caused a partial contraction of worms but worms continued moving for over 2 minutes. Worms exposed to 10~2M atropine for 30 seconds then praziquantel (3 x 10"6M) ceased movement within 30 seconds. If dropped into distilled water adult worms rapidly paralyse. In 0.9 % sodium chloride worms remain moving over an extended period. An equivalent solution of potassium chloride more rapidly paralysed worms but a solution of calcium chloride (.078M) produced a fairly rapid paralysis. By contrast an equivalent solution of magnesium chloride caused worms to gradually relax and cease movement. Worms maintained in the magnesium saline for 5 minutes contracted when treated with praziquantel but gradually relaxed again. Worms maintained in a sodium, potassium chloride saline (NaCl .137M, KC1, 0.0027M) with or without 2mMEDTA for 20 minutes only contracted relatively weakly to praziquantel, those in EDTA responding less than those without EDTA. Three minutes after treatment with praziquantel worms were quite relaxed and some showed sluggish movements. 32
Effect of praziquantel on Schistosoma mansoni
DISCUSSION At similar concentrations the effect of praziquantel is more marked on adult S. mansoni than on larval stages. The daughter sporocysts are resistant to exposure of snails for 24 hours to 3 x 10 5M drug and the effects of short exposures of cercariae to the drug is fully reversible as judged by infectivity tests. The rapid contraction of cercariae in praziquantel with subsequent tachyphylactic type of response, as described for certain nematodes treated with levamisole, (Coles et ah, 1975), could possibly be due to an effect on the nervous system. However the shrivelling of cercariae treated with praziquantel in osmotically concentrated solutions suggests praziquantel affects the ionic or osmotic regulation of the cercariae. Experiments with adult worms confirm this latter interpretation. Four drugs known to affect the nervous system of helminths failed to prevent praziquantel induced contractions, suggesting the drug acts directly on the parasite musculature. Increased calcium ion concentrations caused schistosomes to contract and reduced calcium ion concentrations prevented or reduced the effect of praziquantel. A situation was reached in calcium free solutions where some movement still occurred after praziquantel treatment. Since praziquantel was not active on a calcium ionophore screen (unpublished observations) it is unlikely to be carrying calcium ions across the cell membrane. More probably praziquantel is opening pores in the membrane permitting a rapid influx of calcium ions, either directly or indirectly through an effect on sodium ions. The effect on ion movement and therefore motility is apparently reversible in cercariae (Andrews, 1978) and to a lesser extent in adult worms. A similar reversibility of paralysis in vitro has been observed with contracted tapeworms washed free of praziquantel (Thomas and Andrews, 1977). Detailed electrophysiological examinations of the effect of praziquantel on S. mansoni confirm that it directly or indirectly affects calcium ion movement in the worm (Bennett, 1977—personal communication). An effect on calcium ions could alter cell permeability and explain the leakage of glucose observed with praziquantel treated Hymenolepis diminuta (Thomas and Andrews, 1977). There seems no need to invoke a direct effect on carbohydrate metabolism to explain the drug's schistosomicidal and cestodicidal activity. Why the drug affects one group of trematodes, but not another (liver flukes) remains unclear. ACKNOWLEDGEMENT I wish to thank Dr. P. Andrews of Bayer AG for a gift of pure praziquantel. REFERENCES ANDREWS, P. (1978) Effect of praziquantel on the free living stages of Schistosoma mansoni. Zietschrift fur Parasitenkunde (in press). COLES, G. C , EAST, J. M. and JENKINS, S. W. (1975) The mechanism of action of the anthelmintic levamisole. General Pharmacology, 6, 309-313. GONNERT, R. and ANDREWS, P. (1977) Praziquantel, a new broad spectrum antischistosomal agent. Zeitschrift fur Parasitenkunde, 52, 129-150. PAX, R., BENNETT, J. L. and FETTERER, R. (1978) A benzodiazepine derivative and praziquantel: effects on musculature of Schistosoma mansoni and S.japonicum. Naunyn-Schmiederberg's Archives of Pharmacology, 304, 309-315. THOMAS, H. and ANDREWS, P. (1977) Praziquantel—a new cestodicide. Pesticide Science, 8, 556-560. THOMAS, H. and ANDREWS, P. (1977) The efficacy of praziquantel against cestodes in animals. Zeitschrift fur Parasitenkunde, 52, 117-127. Accepted1) February, 1978.
33
