Pestic. Sci. 1977, 8, 556-560

Praziquantel-A New Cestocidea

Herbert Thomas and Peter Andrews

Bayer Pharma-Forschungszenirum, Instirut fur Chemotherapie, 56 Wiippertal-1, Federal Republic of Gerniany

(Manuscript received 22 October 1976)

Praziquantel is a new chemotherapeutic agent with high activity against juvenile and adult cestodes. The juvenile cestodes tested include Hymenolepis nana, Cysticercus fasciolaris, C . pisiformis, C . tenuicollis, C. bovis. The fully effective doses range from 25-100 mg/kg host. All adult tapeworms tested (Taenia pisiforrnis, T. hydatigena, T. ovis, T. taenaiformis, Dipylidium caninurn, Mesocestoides corti, Echinococcus multilocularis, E. granulosus) of dogs and cats and several Moniezia species of sheep were killed by doses up to 5 mg/kg host. Praziquantel is effective in vitro at very low concentrations. First studies on the mechanism of action in H. diminuta have shown that praziquantel acts on the carbohydrate metabolism of the parasite.

1. Introduction

During the last 20 years new drugs such as niclosamide (I), bunamidine (11) and nitroscanate (111) have been developed for the treatment of tapeworm infections in man and animals. These drugs, however, did not solve all the problems. Therefore, we concentrated on developing a drug with which it would be possible to treat tapeworm infections and at the same time would be well tolerated by the host. These efforts resulted in praziquantel (EMBAY 8440: “Droncit”, registered trade mark of Bayer AG, Leverkusen), which was developed in cooperation by Bayer and E. Merck. Prazi- quantel, 2-cyclohexylcarbonyl-1,3,4,6,7,11 b-hexahydro-2,11 -pyrazino[2,l-a]isoquinolin-4-one (IV), a new type of acylated heterocyclic compound.

Cl OH I I 3N=C -NHC,H,

k

Presented at a symposium Human und animul endoparasites on 22 October 1976 organised jointly by the Pesti- cides and Fine Chemicals Groups, Society of Chemical Industry.

556

Praziquantel-a new cestoclde 551

Preliminary reports on its efficacy against larval and intestinal cestodes have already been g i ~ e n , l - ~ and detailed results are in the press.5 This presentation summarises the results obtained with praziquantel against larval and intestinal tapeworms in mammals and describes the mechanism of action on adult cestodes.

2. Activity against immature infections

The effect of praziquantel against larval stages was tested in mice infected with Hymenolepsis nana cysticercoids and Cysticereus fasciolaris, in rabbits infected with C. pisiformis, in sheep infected with C. tenuicollis and in cattle infected with C. bovis (Table 1). For all these trials experimentally infected animals were used except those with Dipylidium caninum in dogs and Moniezia spp. as well as C. tenuicollis in sheep.

Table 1. Praziquantel-activity against infectious larvae of cestodes

Minimum effective dose

_ _ _ _ _ _ Parasite Host Application (mg/kg) Activity"

Hymenolepis nana-rystirereoids Mouse 1 x oral 25 Cystirercus fasciolaris Mouse 5 x oral 50

25 1 x oral 250

100 5 x subcutaneous 25

10 1 x subcutaneous 250

50 C. pisifornris Rabbit 5 x subcutaneous 25

1 x subcutaneous 50

100 100 63

100 0

100 40

100 25

100 b

C. tenuicollis

C. 60 vis

Sheep 5 x oral

1 x oral Cattle 4 x oral

1 x oral

50 100 10 52 50 100

10-50 100 50 100

(I Parasite reduction (%). Sterilising.

Against C. pisiformis the lowest dose of 25 mg/kg rabbit, given on 5 consecutive days, killed all larvae. A single injection of 50 mg/kg resulted in a sterilising effect, i.e. the larvae did not show any morphological changes but they had lost their ability to infect dogs.

In sheep a single dose of 50 mg/kg was fully effective against C. tenuicollis but 5 x 10 mg/kg on consecutive days killed only about half the larvae. In cattle infected 18 weeks before treatment no live larvae of C. bovis were found 4 weeks after dosing with 4 x 10-50 mg/kg or 1 x 50 mg/kg.

The effect of praziquantel against younger larval stages was examined in mice using H. nana and Taenia taeniaeformis larvae. Against 24- and 48-h-old larvae of H. nana only a 25-73 % effect could be obtained with a dose of 500 mg/kg host. At this age the larvae do not yet show any organ differ- entiation: they consist of a solid cell mass. As their development proceeds they become more and more susceptible to praziquantel. At about 70 h after infection the last step of the differentiation of the sucker and of the scolex begins. At this time the larvae were almost as susceptible to praziquantel as the infectious 96-h-old larvae that were killed by a single oral dose of 25 mg/kg host.

In mice the efficacy of praziquantel against C. fasciolaris also increased with proceeding develop- ment. One- and four-week-old larvae were not yet susceptible to treatment. After the evagination of

558 H. Thomas and P. Andrews

the larvae and at the beginning of the differentiation of the pseudostrobila, 6-7 weeks after infection, the larvae became susceptible to praziquantel. The still older, infectious larvae were more susceptible than the 6- to 7-week old ones. Five doses of 25 mg/kg host were 100% effective on infectious larvae in comparison to 5 x 250 mg/kg with 7-week old larvae.

3. Activity against mature infections

The effect of praziquantel against mature tapeworms was studied in mice, rats, cats, dogs and sheep (Table 2). The results prove that praziquantel is effective on all tapeworm species of dogs and cats tested in doses of 0.5-5 mg/kg host. In sheep praziquantel had a 100% effect at doses ranging from 2.5-5.0 mg/kg against Moniezia species. The efficacy of praziquantel on cestodes in the bile duct was studied in mice on H . microstoma. A complete elimination was obtained after a single oral or subcutaneous administration of 5 or 10 mg/kg, respectively.

Table 2. Praziquantel-efficacy against tapeworms in dogs, cats, rats and mice

Parasite - -___

Hymenolepis naira

H. diminuta

Taenia pisiformis

T. Iiydatigena T. taeniaeformis Dipylidium caninuni Dipylidium caninum

Mesocestoides rorti Echinococcus multilocularis

Host

Mouse

Rat

Efficacy ____ _______ Oral Subcutaneous dose dose

(mgikg) Activity" (mgikg) Activity"

25 100 10 98 10 100 2 . 5 100 1 66 0 . 5 100 0 .25 92 0 . 5 100 1.25 100 0.31 94 0 . 5 100 2 . 5 100 2 . 5 100 I 75 1 7s 5 100 2 . 5 > 99 1 100 1 100

0 . 5 89

- -

Parasite reduction (%).

Praziquantel is effective at extremely low concentrations if it comes into contact with the parasite. Thus injection of praziquantel(2 pg), equivalent to a dose of only 0.08 mg/kg, into the common bile duct of mice infected with H. microstoma reduced the number of parasites by 95 % at dissection two days after treatment. The same effect on parasite reduction could be obtained with an oral dose of 2.5 mg/kg.

There were no marked differences in the susceptibility to praziquantel between adults and juveniles of H . nana, H. diminuta and Mesocestoides corti. Such a difference was found in T. pisi- formis and Echinococcus multilocularis. However, a dose of 5 mg/kg was sufficient to eliminate all stages of dog tape worms that have been investigated.

In mice, it was demonstrated using adult H . nana that age, sex, strain and intensity of infestation of the host had no influence on the efficacy of praziquantel. Although such systematic studies are not available for the other cestode species, the studies carried out by other investigators indicate that these findings are also valid for the other tapeworm species.

Praziquantel-a new cestocide 559

4. I n vitro experiments

4.1. Mode of action Praziquantel is also effective on tapeworms in vitro. At very low concentrations (1.&10 pgllitre) it stimulated motility in H. diminrrta and impaired the proper functioning of the suckers. At concentra- tions of 10-100 pg/litre and above, it caused a very strong contraction of the entire strobila. At a concentration of 1 mg/litre this contraction happened instantaneously on contact of the worm with the drug solution. This very pronounced in-vitro effect of praziquantel on tapeworms and the rapid onset of action on the parasite after treatment of the host both justify the assumption that prazi- quantel itself is the active agent rather than its metabolites.

Our studies of the mechanism of action of praziquantel on cestodes are still far from complete. To date they have mainly dealt with the parasites’ carbohydrate metabolism. In a typical experiment 3 anterior portions of H. dirninuta of 15-25 mg fresh weight each were incubated in 1-2 ml of Hanks solution or Krebs-Ringer hydrogen carbonate solution. Incubation in a solution containing 5 mM glucose resulted in a decrease of glucose in the medium because of the uptake of glucose by the tapeworms. Addition of praziquantel to the incubation medium resulted in a marked decrease in glucose uptake. The rate of glocuse uptake was about halved in the presence of 100 pg praziquantel/ litre. However, even in high concentrations of praziquantel (3 mg/litre) there was still more glucose taken up than in the heat-killed control group.

Praziquantel further induced an increase in the amount of lactate excreted into the medium over that found in the controls. Again, a concentration of 100 pg/litre was sufficient to give a significant effect. Praziquantel induced an increase in lactate excretion both in media containing glucose and those free of glucose, indicating that the lactate excreted was formed from endogenous carbohy- drates, presumably glycogen. A decrease in glycogen content of the worms was difficult to verify in incubation experiments. However, if rats harbouring 11. diminuta were orally treated with 25mg praziquantel/kg, there was a marked drop of 20-50% in the glycogen content of the worms retrieved after 2 h from the intestine of treated as compared to placebo-treated rats.

A further effect of praziquantel on the tapeworm could be. observed on incubation of H. diminuta in glucose-free media. Praziquantel rendered the tegument permeable to glucose. Therefore, glucose was leaking from the parasite into the medium. This leakage of glucose is a specific effect of praziquantel. Neither live nor heat-killed untreated worms lost glucose to the medium.

In future experiments we will try to determine the concentration of various metabolites in treated worms in the hope that this will produce further evidence for the mechanism of action of prazi- quantel.

4.2. Reversible nature of in-vitro action Our incubation experiments have also shown a phenomenon that was quite unexpected from all in- vitro experiments. This is thereversibility, at least in principle, of the deleterious effects of praziquan- tel. If worms were incubated in media containing praziquantel for 15 min and then placed into drug- free media one observed a restitution of impermeability of the tegument to glucose. The praziquan- tel-induced contraction slowly receded and the worms regained their motility. Glucose uptake also recovered after an initial incubation in praziquantel solution.

One reason for the apparent lack in reversibility of drug action in vivo may be due to another effect on the tapeworms. The tegument is readily attacked by proteolytic enzymes in the presence of praziquantel. Q vivo therefore, the tapeworm may well be irreparably damaged by proteolysis as well as by dislocation in the intestine before the effect of praziquantel wears off.

5. Conclusion

With praziquantel, a drug is now available which has an extremely high therapeutic index. Under most varying laboratory and field conditions, single doses were reliably effective on all species and age groups of cestodes tested in mice, rats, cats, dogs and sheep. In addition to the effect on Taeniu

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560 H. Thomas and P. Andrews

species, D. caninum and Mesocestoides corti, the reliable effect on Echinococcus granulosus and E. multilocularis is of particular interest. Thus, praziquantel is extremely well suited for the eradication of Echinococcus spp. in dogs.

References I. Thomas, H.; Gonnert, R.; Pohlke, R.; Seubert, J. Pror. 71h in!. Conf. wld Ass. Parasi!.Thessaloniki, 1975. 2. Thomas, H.; Gonnert, R.; Pohlke, R.; Seubert, J. Pror. 71h int. Conf. wld Ass. Parasit. Thessaloniki 1975. 3. Thomas, H.; Gonnert, R.; Pohlke, R.; Seubert, J. 2nd European Multi-Colloquy of Parasi!ology, Trogir 1975. 4. Thomas, H.; Gonnert, R.; Pohlke, R.; Seubert, J. 2nd European Multi-Co1loqu.v of Parasitolog.~, Trogir 1915. 5. Thomas, H.; Gonnert, R. Res. Vet. Sci. 1977.