Journal of Helmlnthology (1975) 49, 205—209
The response of Schistosoma mansoni maintained in vitro to schistosomicidal compounds G. C. COLES Imperial Chemical Industries Ltd., Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire SK10 4717, England
ABSTRACT The activity is reported of certain clinical and experimental schistosornicides against schistosomula, 3 week old, and adult worms of Schistosoma mansoni maintained in vitro. Since the test method is not sufficiently selective for use with random compounds, it is concluded that meaningful screening for potential schistosomicides cannot at present by carried out in vitro.
The limited quantity of chemical available or the large amount of work involved in screening in mammals may necessitate the testing of novel chemicals against schistosomes maintained in vitro. A number of papers have appeared in which the activity of individual drugs against schistosomes maintained in vitro has been reported (e.g. Foster and Cheetham, 1973; Lee, 1972; Lee and Michaels, 1968; Magzoub, 1972; McLure and Ercoli, 1971; Pellegrino and Maria, 1966) but there has been little attempt to see whether the methods used are applicable to screening. Young worms have not been used, although the possibility of using schistosomula in chemotherapeutic studies has been raised (Tiba et al.t 1974). The present study was undertaken to compare the activity of a number of known schistosomicides and related compounds against schistosomula, three-week-old worms and adults to see which of the stages, if any, could be used for routine screening in vitro. MATERIALS AND METHODS S. mansoni (Wellcome strain) was maintained by standard procedures in Biomphalaria glabrata and white mice (Alderley Park strain). Schistosomula were prepared by the method of Clegg and Smithers (1972), three-week-old worms by perfusion of mice infected with 2,000 cercariae with Tyrode's solution containing heparin, and adult worm pairs by sterile dissection. The younger stages were washed in sterile Earle's lactalbumin and all stages were cultured in Earle's lactalbumin/new bora calf serum (1:1) containing antibiotics as used for culture of schistosomula by Clegg and Smithers (1972). Worms were placed in 2 ml of medium in multicompartment sterile dishes, and placed in air-tight containers gassed with 95 per cent air plus 5 per cent COZ and maintained at 37 °C. The lower concentration of carbon dioxide in the gas phase, 5 per cent compared with 8 per cent recommended by Clegg and Smithers, was chosen as it was already in use for other culture procedures in the laboratory and it did not appear to adversely affect the survival of the worms. Approximately 20 schistosomula, six three-week-old worms or two adult pairs were placed in each compartment. Compounds to be tested were dissolved in dimethyl sulphoxide, a maximum volume of 10/il ot solvent being added. A similar volume of dimethyl sulphoxide was always added to control worms, but did not affect the survival of the worms in the amounts used. 205
1M, 5D 2A, 1M.3D
2A, 1M, 3D 5A, ID
6A
6A
1.0 10
1.0 30 60
0.5 1.0 30 60
Lucanthonc
Oxamniquine
Metrifonate
1A, 5D 6D
1M, 5D
4A,2D
5A, 1M
1.0 10
Hycanthone
3A, 1M, 2D 1A.5D
6D
2A, 1M, 3D
6A
1.0 10 20
Niridazolc
6A 2A, INf, 3D
6D
4A.2D 6D
72h
5A, 1M 3A, 3D 6D
48h
0.5 1.0 2.5
24h
Schistosomula
KSbtartrate
Drug
cone, p.p.m.
6A 6A
6A 6A
6A
6A
6A 6A
6A 6A
24h
6A 4A.2M
6A 2A,4M
5A, 1M
4A,2M
6A 4A,2M
4A,2D 2A,4D
48h
6A 1M, 5D
6A 4M, 2D
1M.5D
6D
2A, 2M, 2D 6D
2A.4D 6D
72h
3 week old worms
3A, 3M
6A
6A
6A
6A 4A.2D
6A 4A,2D
24h
1A, 1M.4D
4M.2D
4A, 1M, ID
6A
4A.2D 6D
1A.5D 6D
48h
adult worms
2M,4D
4M.2D
6D
1M,5D
2A.4D
6D
72h
The activity of clinical schistosomicides against S. mansoni maintained in vitro. All results are given as activity of worms observed in six replicates. (A, worms active: M, very slight movement; D, no movement)
TABLE 1
8
P
j
o
to
6A
6A
1.0 10 20
1.0 20
1.0 30 60
1.0 30 60
Chloxyle
dchydrocmetinc hydrochloridc
Wellcome 62T59
Wellcome 153C51
4A.2D
6A
5A, 1M 1A.5D
0.5 1.0
SchistocideT102
6A 3A.3D
0.5 1.0 60
24h
Acinitrazolc
Compound
cone, p.pjn.
5A, 1M
2M.4D
2A,4D
4A.2D
2A, 1M, 3D 6D
1 A, 1M, 4D 6D
48h
Schistosomula
2A.4D
6D
6D
6D
6D
6D
72h
6A 6A
6A 6A
6A
6A
,
4A.2M
6A
24h
6A 1A,5M
6A 6A
6A
6A
1A, 1M, 4D
5A, ID
48h 72h
6A 1M.5D
6A 6D
6A
6A
6D
4A.2D
3 week old worms
3A,3M
1A.5M
6A
6A 6A
6A
6A
24h
3M.3D
4M.2D
3A.3M
6A 2A.3M, ID
2A.2M, 2D
4A,2D
48h
72h
2M.4D
2M.4D
3M,3D
3A, 1M, 2D 1M, 5D
6D
2A,4D
Adult worms
The activity of experimental schistosomicides against S. mansoni maintained in vitro. All results are given as activity of worms observed in six replicates. (A, worms active; M, very slight movement; D, no movement)
TABLE 2
1 I
cu
o!
o
8
G. C. COLES
Acinltrazole
fl— N
NHC0CH 3
Dehydroemetine hydrochloride
Schlstocide T102
Wellcome 62T59
Chloxyle
Wellcome 153C51 2HC1 FIG. 1. Structure of experimental schistosomiddes in Table 2.
The activity of the worms was observed after 24, 48 and 72 hours on an inverted microscope. Unless all adult and three-week-old worms or at least 90 per cent of schistosomula were alive and active in the controls, the results of the tests were not recorded. Worms under test were classified as either active, showing very slight movement, or dead (no visible movement). RESULTS The results of the tests with six clinical schistosomicides and six experimental schistosomicides are given in tables 1 and 2. Schistosomula were always more sensitive to drugs than three-week-old worms and adults. In several cases, but not all, three-week-old worms were more resistant than adult worms to drugs. From the tables it can be seen that in most tests all the compounds examined would be detected by using schistosomula and 1 ppm of chemical. Therefore computer selected samples of recently synthesised compounds (i.e. "random" selection) were tested at this concentration against schistosomula. Of 400 compounds tested, 80 (20 per cent) killed schistosomula. As this was considered to be too high a percentage activity, further selections of compounds were tested at 20 ppm against three-week-old and adult woims. Although this would be too low a concentration to detect all compounds listed in Tables 1 and 2 the rate of kill for 200 compounds tested against three-week-old worms was 10 per cent and for 125 compounds tested against adult worms, 27 per cent. DISCUSSION It is clear from the results of testing of randomly selected chemicals that screening of chemicals in vitro produces too many positive compounds if the screening is operated at levels that will detect known active drugs. Since the requirements of an effective screen are that it will detect known drugs and be selective, the present methods are not satisfactory 208
Schistosomicides against S. mansoni in vitro
for looking for schistosomicides. It is recognized that the presence of protein in the medium decreases the activity of many compounds, as described for hycanthone (Lee, 1972), and this will probably explain why niridazole appeared less active in the present screen than in the experiments of Lee and Michael (1968). But there is no reason to believe that the absence of serum would make the test more selective for schistosomicides. Schistosomula appear more sensitive to drugs than adult worms, but the reverse appears to be usually the case in vivo (Coles. 1973). This has been examined in detail for potassium antimony tartrate (Coles and Chappell, in preparation), and the possibility was raised that one role of some drugs might be so to affect the surface of the schistosome that the host can recognize the presence of foreign protein and destroy it. If such a situation occurs then it is obvious that an in vitro screen using normal serum would be inappropriate. The cost and problems of using immune sera, possibly containing blood cells, would render the system impractical for routine use. At present meaningful screens run in vitro for schistosomicides do not appear a practical possibility. ACKNOWLEDGEMENTS I am grateful to Miss J. Selby for technical assistance and to Burroughs Wellcome & Co., Ciba U.K., Pfizer and Sterling-Winthrop for gifts of drugs.
REFERENCES CLEGG, J. A. and SMITHERS, S. R. (1972) The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. International Journal for Parasitology, 2,79-98. COLES, G. C. (1973) The metabolism of schistosomes: a review. InternationalJournal of Biochemistry, 4, 319-337. FOSTER, R. and CHEETHAM, B. (1973) Studies with the schistosomicide oxamniquine (UK-4271) 1. Activity in rodents and in vitro. Transactions of the Royal Society of tropical Medicine and Hygiene, 67, 674-684. LEE, H. G. (1972) Aspects of thioxanthone on Schistosoma mansoni in mice and in vitro. Bulletin of the World Health Organisation, 46, 397-402. LEE, H. G. and MICHAELS, R. M. (1968) In vitro and in vivo effects of selected metabolic inhibitors and chemothcrapeutic agents on adults and egg development of Schistosoma mansoni. Experimental Parasitology, 22, 256-263. MAGZOUB, B. (1972) In vitro effects of graded concentrations of niridazole (Ambilhar) and stibocaptate (Astiban) on the glycolytic pathway and motility of the Egyptian strain of Schistosoma mansoni. Comparative and General Pharmacology, 3, 401-409. McLURE, M. T. and ERCOLI, N. (1971) In vitro comparison of the anti-schistosomal effect of trivalcnt antimonials and their inhibition by the organic radical of stibophen. Ada cientifica venezolana, 22, 207-208. PELLEGRINO, J. and DE MARIA, M. (1966) In vitro cercaricidal activity of schistosomicides. Journal of Parasitology, 52, 617. TIBA, Y., HOLANDA, J. C , RAMALHO-PINTO, F. J., GAZZINELLI, G. and PELLEGRINO, J. (1974) Schistosomula (Schistosoma mansoni) obtained in vitro: viability in culture and infectivity for mice. Transactions of the Royal Society of tropical Medicine and Hygiene, 68, 72. Accepted 9 July 1975
209
The response of Schistosoma mansoni maintained in vitro to schistosomicidal compounds G. C. COLES Imperial Chemical Industries Ltd., Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire SK10 4717, England
ABSTRACT The activity is reported of certain clinical and experimental schistosornicides against schistosomula, 3 week old, and adult worms of Schistosoma mansoni maintained in vitro. Since the test method is not sufficiently selective for use with random compounds, it is concluded that meaningful screening for potential schistosomicides cannot at present by carried out in vitro.
The limited quantity of chemical available or the large amount of work involved in screening in mammals may necessitate the testing of novel chemicals against schistosomes maintained in vitro. A number of papers have appeared in which the activity of individual drugs against schistosomes maintained in vitro has been reported (e.g. Foster and Cheetham, 1973; Lee, 1972; Lee and Michaels, 1968; Magzoub, 1972; McLure and Ercoli, 1971; Pellegrino and Maria, 1966) but there has been little attempt to see whether the methods used are applicable to screening. Young worms have not been used, although the possibility of using schistosomula in chemotherapeutic studies has been raised (Tiba et al.t 1974). The present study was undertaken to compare the activity of a number of known schistosomicides and related compounds against schistosomula, three-week-old worms and adults to see which of the stages, if any, could be used for routine screening in vitro. MATERIALS AND METHODS S. mansoni (Wellcome strain) was maintained by standard procedures in Biomphalaria glabrata and white mice (Alderley Park strain). Schistosomula were prepared by the method of Clegg and Smithers (1972), three-week-old worms by perfusion of mice infected with 2,000 cercariae with Tyrode's solution containing heparin, and adult worm pairs by sterile dissection. The younger stages were washed in sterile Earle's lactalbumin and all stages were cultured in Earle's lactalbumin/new bora calf serum (1:1) containing antibiotics as used for culture of schistosomula by Clegg and Smithers (1972). Worms were placed in 2 ml of medium in multicompartment sterile dishes, and placed in air-tight containers gassed with 95 per cent air plus 5 per cent COZ and maintained at 37 °C. The lower concentration of carbon dioxide in the gas phase, 5 per cent compared with 8 per cent recommended by Clegg and Smithers, was chosen as it was already in use for other culture procedures in the laboratory and it did not appear to adversely affect the survival of the worms. Approximately 20 schistosomula, six three-week-old worms or two adult pairs were placed in each compartment. Compounds to be tested were dissolved in dimethyl sulphoxide, a maximum volume of 10/il ot solvent being added. A similar volume of dimethyl sulphoxide was always added to control worms, but did not affect the survival of the worms in the amounts used. 205
1M, 5D 2A, 1M.3D
2A, 1M, 3D 5A, ID
6A
6A
1.0 10
1.0 30 60
0.5 1.0 30 60
Lucanthonc
Oxamniquine
Metrifonate
1A, 5D 6D
1M, 5D
4A,2D
5A, 1M
1.0 10
Hycanthone
3A, 1M, 2D 1A.5D
6D
2A, 1M, 3D
6A
1.0 10 20
Niridazolc
6A 2A, INf, 3D
6D
4A.2D 6D
72h
5A, 1M 3A, 3D 6D
48h
0.5 1.0 2.5
24h
Schistosomula
KSbtartrate
Drug
cone, p.p.m.
6A 6A
6A 6A
6A
6A
6A 6A
6A 6A
24h
6A 4A.2M
6A 2A,4M
5A, 1M
4A,2M
6A 4A,2M
4A,2D 2A,4D
48h
6A 1M, 5D
6A 4M, 2D
1M.5D
6D
2A, 2M, 2D 6D
2A.4D 6D
72h
3 week old worms
3A, 3M
6A
6A
6A
6A 4A.2D
6A 4A,2D
24h
1A, 1M.4D
4M.2D
4A, 1M, ID
6A
4A.2D 6D
1A.5D 6D
48h
adult worms
2M,4D
4M.2D
6D
1M,5D
2A.4D
6D
72h
The activity of clinical schistosomicides against S. mansoni maintained in vitro. All results are given as activity of worms observed in six replicates. (A, worms active: M, very slight movement; D, no movement)
TABLE 1
8
P
j
o
to
6A
6A
1.0 10 20
1.0 20
1.0 30 60
1.0 30 60
Chloxyle
dchydrocmetinc hydrochloridc
Wellcome 62T59
Wellcome 153C51
4A.2D
6A
5A, 1M 1A.5D
0.5 1.0
SchistocideT102
6A 3A.3D
0.5 1.0 60
24h
Acinitrazolc
Compound
cone, p.pjn.
5A, 1M
2M.4D
2A,4D
4A.2D
2A, 1M, 3D 6D
1 A, 1M, 4D 6D
48h
Schistosomula
2A.4D
6D
6D
6D
6D
6D
72h
6A 6A
6A 6A
6A
6A
,
4A.2M
6A
24h
6A 1A,5M
6A 6A
6A
6A
1A, 1M, 4D
5A, ID
48h 72h
6A 1M.5D
6A 6D
6A
6A
6D
4A.2D
3 week old worms
3A,3M
1A.5M
6A
6A 6A
6A
6A
24h
3M.3D
4M.2D
3A.3M
6A 2A.3M, ID
2A.2M, 2D
4A,2D
48h
72h
2M.4D
2M.4D
3M,3D
3A, 1M, 2D 1M, 5D
6D
2A,4D
Adult worms
The activity of experimental schistosomicides against S. mansoni maintained in vitro. All results are given as activity of worms observed in six replicates. (A, worms active; M, very slight movement; D, no movement)
TABLE 2
1 I
cu
o!
o
8
G. C. COLES
Acinltrazole
fl— N
NHC0CH 3
Dehydroemetine hydrochloride
Schlstocide T102
Wellcome 62T59
Chloxyle
Wellcome 153C51 2HC1 FIG. 1. Structure of experimental schistosomiddes in Table 2.
The activity of the worms was observed after 24, 48 and 72 hours on an inverted microscope. Unless all adult and three-week-old worms or at least 90 per cent of schistosomula were alive and active in the controls, the results of the tests were not recorded. Worms under test were classified as either active, showing very slight movement, or dead (no visible movement). RESULTS The results of the tests with six clinical schistosomicides and six experimental schistosomicides are given in tables 1 and 2. Schistosomula were always more sensitive to drugs than three-week-old worms and adults. In several cases, but not all, three-week-old worms were more resistant than adult worms to drugs. From the tables it can be seen that in most tests all the compounds examined would be detected by using schistosomula and 1 ppm of chemical. Therefore computer selected samples of recently synthesised compounds (i.e. "random" selection) were tested at this concentration against schistosomula. Of 400 compounds tested, 80 (20 per cent) killed schistosomula. As this was considered to be too high a percentage activity, further selections of compounds were tested at 20 ppm against three-week-old and adult woims. Although this would be too low a concentration to detect all compounds listed in Tables 1 and 2 the rate of kill for 200 compounds tested against three-week-old worms was 10 per cent and for 125 compounds tested against adult worms, 27 per cent. DISCUSSION It is clear from the results of testing of randomly selected chemicals that screening of chemicals in vitro produces too many positive compounds if the screening is operated at levels that will detect known active drugs. Since the requirements of an effective screen are that it will detect known drugs and be selective, the present methods are not satisfactory 208
Schistosomicides against S. mansoni in vitro
for looking for schistosomicides. It is recognized that the presence of protein in the medium decreases the activity of many compounds, as described for hycanthone (Lee, 1972), and this will probably explain why niridazole appeared less active in the present screen than in the experiments of Lee and Michael (1968). But there is no reason to believe that the absence of serum would make the test more selective for schistosomicides. Schistosomula appear more sensitive to drugs than adult worms, but the reverse appears to be usually the case in vivo (Coles. 1973). This has been examined in detail for potassium antimony tartrate (Coles and Chappell, in preparation), and the possibility was raised that one role of some drugs might be so to affect the surface of the schistosome that the host can recognize the presence of foreign protein and destroy it. If such a situation occurs then it is obvious that an in vitro screen using normal serum would be inappropriate. The cost and problems of using immune sera, possibly containing blood cells, would render the system impractical for routine use. At present meaningful screens run in vitro for schistosomicides do not appear a practical possibility. ACKNOWLEDGEMENTS I am grateful to Miss J. Selby for technical assistance and to Burroughs Wellcome & Co., Ciba U.K., Pfizer and Sterling-Winthrop for gifts of drugs.
REFERENCES CLEGG, J. A. and SMITHERS, S. R. (1972) The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. International Journal for Parasitology, 2,79-98. COLES, G. C. (1973) The metabolism of schistosomes: a review. InternationalJournal of Biochemistry, 4, 319-337. FOSTER, R. and CHEETHAM, B. (1973) Studies with the schistosomicide oxamniquine (UK-4271) 1. Activity in rodents and in vitro. Transactions of the Royal Society of tropical Medicine and Hygiene, 67, 674-684. LEE, H. G. (1972) Aspects of thioxanthone on Schistosoma mansoni in mice and in vitro. Bulletin of the World Health Organisation, 46, 397-402. LEE, H. G. and MICHAELS, R. M. (1968) In vitro and in vivo effects of selected metabolic inhibitors and chemothcrapeutic agents on adults and egg development of Schistosoma mansoni. Experimental Parasitology, 22, 256-263. MAGZOUB, B. (1972) In vitro effects of graded concentrations of niridazole (Ambilhar) and stibocaptate (Astiban) on the glycolytic pathway and motility of the Egyptian strain of Schistosoma mansoni. Comparative and General Pharmacology, 3, 401-409. McLURE, M. T. and ERCOLI, N. (1971) In vitro comparison of the anti-schistosomal effect of trivalcnt antimonials and their inhibition by the organic radical of stibophen. Ada cientifica venezolana, 22, 207-208. PELLEGRINO, J. and DE MARIA, M. (1966) In vitro cercaricidal activity of schistosomicides. Journal of Parasitology, 52, 617. TIBA, Y., HOLANDA, J. C , RAMALHO-PINTO, F. J., GAZZINELLI, G. and PELLEGRINO, J. (1974) Schistosomula (Schistosoma mansoni) obtained in vitro: viability in culture and infectivity for mice. Transactions of the Royal Society of tropical Medicine and Hygiene, 68, 72. Accepted 9 July 1975
209
