Zeitschrift f0r

Parasitenkunde

Z. Parasitenkd. 58, 169 174(1979)

Parasitology Research

9 by Springer-Verlag 1979

Schistosoma mansoni:

Activity Responses in vitro to Praziquantel C.J. C h a v a s s e 1, M . C . B r o w n

2

a n d D . R . Bell 1

1 Department of Tropical Medicine, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK 2 Bioengineering and Medical Physics Unit, University of Liverpool, Liverpool L69 3BX, UK

Summary. T h e effects o f p r a z i q u a n t e l , a novel a n t i s c h i s t o s o m a l c o m p o u n d , on the activity o f a d u l t Schistosoma mansoni ( L i v e r p o o l strain) in vitro were investigated. W o r m activity was m o d i f i e d at all c o n c e n t r a t i o n s o f p r a z i q u a n t e l . H i g h c o n c e n t r a t i o n s ( a b o v e 0.5 gg/ml) p r o d u c e d r a p i d paralysis, whilst low c o n c e n t r a t i o n s o f p r a z i q u a n t e l s t i m u l a t e d w o r m activity. It is c o n c l u d e d t h a t such activity m o d i f i c a t i o n c o u l d l e a d to w o r m d i s p l a c e m e n t in vivo.

Introduction P r a z i q u a n t e l ( E m b a y 8440) ( P Z Q ) is a novel b r o a d s p e c t r u m a n t i s c h i s t o s o m a l a g e n t ( A n d r e w s , 1977; G 6 n n e r t a n d A n d r e w s , 1977; J a m e s et al., 1977; Pellegrino et al., 1977; W e b b e a n d James, 1977). T h e r a p e u t i c doses halt egg p r o d u c tion a n d cause a shift o f w o r m s to the liver where they are s u b s e q u e n t l y d e s t r o y e d by the host. This p a p e r r e p o r t s the activity r e s p o n s e in vitro o f a d u l t Schistosoma mansoni to P Z Q .

Materials and Methods The activity of single adult S. mansoni worms (Liverpool strain) was monitored in vitro by the ultrasonic method of Brown et al. (1978 b). Worms were obtained from OLAC MFI female white mice 7-10 weeks after infection, and placed in a continuous flow of 50% serum-based culture medium as described previously (Chavasse et al., 1978). Activity data were recorded for ten hours and processed according to Brown et al. (1978a). Praziquantel (Bayer Pt t/77) was dissolved in ethanol, 10 mg/ml, then diluted 1 in 10 in culture medium, before being added to the desired final concentration in 50 ml of medium in a reservoir bottle. The first five-hour period was used to establish a reference activity level, before the drug was added. The concentrations of praziquantel tested ranged from 10 gg/ml to 0.01 gg/ml.

0044-3255/79/0058/0169/$01.20

C.J. Chavasse et al.

170

Drug adde( PZQ 10~ug/m[ 10expts. L

PZQ 1,ug/m[ 6expts.

PZO 0,5ug/m[ 12expts. ,-~,,~_.._.%f_v ^ f " - - . ~

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PZQ 0,2JJg/ml 10expts.

PZQ 0,1 ,ug/ml 11expts. "~"m-~,./~v.. ~ ~ v , - X ~-

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PZQ 0,01 #g/mr 11expts.

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Control. 22 expts. o

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Time- hours Fig. 1. Schistosoma mansoni. Averaged activity records for the stated number of experiments at different praziquantel concentrations. The zero 'base line' for each graph is drawn through the mean activity value for the period before PZQ addition, and represents the reference activity level

Results The results are summarised in Figs. 1 and 2 and Table 1. Figure 1 shows the average activity response for all the experiments performed at each concentration. Table 1 provides some numerical details to elucidate aspects of the activity pattern which may not be clear in Fig. 1. Figure 2 shows the variation in the mean activity change for each case shown in Fig. 1, and also indicates that the shift in mean activity showed more variation in the drug tests than in either the controls or in tests to estimate the effects of the drug solvent (ethanol) on the results.

Schistosoma mansoni Response to Praziquantel

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-1,5' Fig. 2. Schistosoma mansoni. Plot (from Table 1) of the mean activity change at each PZQ concentration to show the variation of this change in each set of tests. The effect of alcohol, at a concentration equivalent to that present in praziquantel solutions of 0.01 gg/ml, is also illustrated Table 1. Activity data: Statistical s u m m a r y PZQ concentration ~tg/mi

Ae

sl

s2

s~

D

Number of experiments

10 1 0.5 0.2 0.1 0.05 0.01 Alcohol

-0,827 - 1.125 -0.316 + 0.093 + 0.244 +0.080 +0.012 + 0.016

0.080 0.057 0.084 0.075 0.073 0.077 0.085 0,076

0.054 0.093 0.089 0.106 0.045 0.048 0.071 0.069

0.512 0.279 0.607 0.518 0.209 0.234 0.241 0.103

128.6 169.9 42.5 11.8 46.9 13.3 1.6 2.6

10 6 12 10 11 14 11 I1

Control

+ 0.034

0,049

0.045

0.136

8.0

22

A~ is the average change in mean activity for the stated number of experiments between the two five-hour culture periods, si and sz are the standard deviation of the mean activity during the first and second periods respectively. D is the normal deviate given by: D=

AY

]/

1 + 1.1/(ni-1)s:~+(n2-1)s~ n1

n~ ~

nt+n2-2

where nl and n2 are the respective n u m b e r of activity records taken from each period, with nx~n2>250.

172

c.J. Chavasseet al.

For the range of drug concentrations tested, we have found three main categories of activity response. Low concentrations caused a slight activity disturbance, intermediate concentrations increased activity, and high concentrations suppressed activity. In the control group, the mean activity differed slightly between the two five-hour culture periods. However, the standard deviation of this change (sz) in the relevant 22 control tests was small, indicating that there was no major disturbance of the activity pattern. The response to PZQ 0.01 gg/ml was a slight activity increase. However, the extent of the disturbance of the individual activity patterns was greater than this result alone would suggest, and this is shown by Fig. 2. Considerable variation in the response to PZQ 0.05 gg/ml was also observed, although at this concentration there was a significant activity increase, sustained for the experimental period. A similar, but greater, activity increase occurred at PZQ 0.1 gg/ml. At 0.2 gg PZQ/ml, a rapid activity increase was followed by a gradual decline to a level similar to that obtained during the reference period. Worm activity was suppressed by PZQ 0.5 gg/ml after an initial period of stimulation, and although recorded activity fell below the reference level, the worms remained active. For 1 and 10 gg PZQ/ml activity was rapidly and completely suppressed. After 1-2 h exposure to 1 gg/ml, the worms showed some signs of regaining activity, but exposure to a stimulant (5-hydroxytryptamine) at the end of the culture period failed to produce any further activity changes.

Discussion

G6nnert and Andrews (1977) reported that a maximum plasma concentration of 0.4 gg PZQ/ml was necessary over a period of several hours for efficacy against schistosomes in vitro. Andrews (1976) found that the lower limit in vitro for detection of worm contraction was 0.01 gg PZQ/ml in Eagles medium. PZQ is rapidly distributed and eliminated in various laboratory animals, although less rapidly in larger animals (Andrews, 1976; Diekmann and Bfihring, 1976; Steiner and Garbe, 1976; Steiner et al., 1976). In large animals a single oral dose equivalent to the total dose given in spaced treatment was equally effective as the spaced treatment in producing parasite reduction, whereas for smaller animals (rodents) multiple dose treatment during one day was the most effective treatment (G6nnert and Andrews, 1977; James et al., 1977; Pellegrino et al., 1977; Webbe and James, 1977). However, even a non-therapeutic single oral dose of 10 mg/kg produced a complete but transient liver shift in S. mansoniinfected mice (Andrews, 1977) and 25 mg/kg in Cebus monkeys halted egg production (Pellegrino et al., 1977). Peak levels of unchanged PZQ in serum in various animals in the range 0.03-0.72 gg/ml 15-30 min after a single oral dose of 10 mg/kg would be expected, with a concentration above 0.01 gg/ml prevailing for a further 4 h (Steiner et al., 1976).

Schistosoma mansoni Response to Praziquantel

173

O u r in vitro results indicate that P Z Q concentrations of this magnitude disturb or slightly stimulate w o r m activity. Such a response in vivo could result in w o r m s failing to maintain their position, and render them m o r e susceptible to being dislodged by the bloodstream. At higher P Z Q concentrations in vitro w o r m s are paralysed, and this would be expected to result in a liver shift in vivo. It is necessary for w o r m s to be retained in the liver for approximately four days after P Z Q treatment for the w o r m s to be destroyed (Andrews, 1977; Pellegrino et al., 1977). O u r results suggest that continued exposure, even to low P Z Q concentrations, could sustain activity modification and hence w o r m displacement. However, w o r m s have been f o u n d dead in situ in the mesenteric vessels o f S. haernatobium-infected primates (James et al., 1977) indicating that a shift to the liver is n o t essential for w o r m destruction. The m o d e o f action o f P Z Q is not fully understood, but it appears to disturb the cation balance o f the parasite (Pax et al., 1978) and this may affect the m e m b r a n e potential o f muscle cells. Activity modification has been shown for w o r m s exposed to 0.01 gg P Z Q / m l in vitro. This m a y be close to the lower resolution limit o f the ultrasonic detection system and m a y a p p r o a c h the lowest concentration of P Z Q which produces activity modification. However, at even lower concentrations (0.001 ~tg/ ml) in vitro P Z Q inhibits egg p r o d u c t i o n (Andrews, 1977). In conclusion, our results indicate that the P Z Q concentrations necessary to paralyse S. mansoni w o r m s in vitro are relatively high c o m p a r e d with anticipated serum levels in man, whereas only low concentrations are necessary to m o d i f y w o r m activity in a way which could cause w o r m displacement in vivo. Acknowledgements. We are grateful to Professor H.M. Oilles for providing facilities for this work

in the Department of Tropical Medicine. Praziquantel was donated by Bayer AG (Wuppertal, Federal Republic of Germany). Financial support was provided by the Edna McConnell Clark Foundation (Grant No. 276-0013).

References Andrews, P. : Pharmacokinetic studies with Droncit in animals using a biological assay. Vet. Med. Rev. 2, 154-165 (1976) Andrews, P. : Praziquantel - a novel schistosomicide. Proceedings of British Society for Parasitology. Parasitology 75, XVII-XVIII (1977) Brown, M.C., Chavasse, C.J., Norman, D.F., Bell, D.R.: Evoked response averaging techniques used to clarify the effect of drugs on the in vitro activity of schistosomes. Med. Biol. Eng. 16, 597-598 (1978a) Brown, M.C., Norman, D.F., Bell, D.R., Chavasse, C.J. : A multichannel ultrasonic activity monitor for in vitro screening of antischistosomal drugs. Med. Biol. Eng. 16, 408-418 (1978b) Chavasse, C.J., Brown, M.C., Bell, D.R.: Short-term effects of oxamniquine on the activity in vitro of Schistosoma mansoni. Ann. Trop. Med. Parasitol. 72, 293-295 (1978) Diekmann, H.W., Biihring, K.U.: The fate of praziquantel in the organism. III. Metabolism in rat, beagle dog and rhesus monkey. Eur. J. Drug Metab. Pharmakin., 2, 107-112 (1976) G6nnert, R., Andrews, P. : Praziquantel, a new broad-spectrum antischistosomal agent. Z. Parasitenkd. 52, 129-150 (1977)

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James, C., Webbe, G., Nelson, G.S. : The susceptibility to praziquantel of Schistosoma haematobium in the baboon (Papio anubis) and of S. japonicum in the vervet monkey (Cercopithecus aethiops). Z. Parasitenkd. 52, 179 194 (1977) Pax, R., Bennett, I.L., Fetterer, R.: A benzodiazepine derivative and praziquantel: Effects on musculature of Schistosoma mansoni and Schistosoma japonicum. Arch. Pharmacol. 304, 309-315 (1978) Pellegrino, J., Lima-Costa, F.F., Carlos, M.A., Mello, R.T. : Experimental chemotherapy of schistosomiasis mansoni. XIII. Activity of praziquantel, an isoquinoline-pyrazino derivative, on mice, hamsters and Cebus monkeys. Z. Parasitenkd. 52, 151-168 (1977) Steiner, K., Garbe, A. : The fate of praziquantel in the organism. II. Distribution in rats. Eur. J. Drug Metab. and Pharmakin., 2, 97-106 (1976) Steiner, K., Garbe, A., Diekmann, H.W., Nowak, H.: The fate of praziquantel in the organism. I. Pharmacokinetics in animals. Eur. J. Drug Metab. and Pharmakin., 2, 85-95 (1976) Webbe, G., James, C. : A comparison of the susceptibility to praziquantel of Schistosoma haematobium, S. japonicum, S. mansoni, S. intercaIatum and S. matthei in hamsters. Z. Parasitenkd, 52, 169 177 (1977) Received August 14, 1978

Schistosoma mansoni: activity responses in vitro to praziquantel.

Zeitschrift f0r Parasitenkunde Z. Parasitenkd. 58, 169 174(1979) Parasitology Research 9 by Springer-Verlag 1979 Schistosoma mansoni: Activity R...
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