Parasitologia Hungarica 3. (Budapest, 1970)

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Parasitologia Hungarica 3. (Budapest, 1970)

Parasit. Hun». H. 147-158.1970

In Vitro Action of Tetramisole on Ascaris suum

Dr.

Anna LENGYEL — Dr. László Z0LÏA1

Department of Parasitology,

National Institute of Public Health, Budapest

Detection of the anti-nematode action of piperazine dérivâtes

is regarded as the first success in the search for anthelmintics

on the basis of biochemical and pharmacological

studies. Following the discovery of the efficacy of diethylcarbamazine

for the therapy of filariosis various salts of piperazine

were shown to be effective for the treatment of ascaridosis.

later findings resulted in drugs specific for ascaridosis

which are s t i l l in use.

The efficacy of piperazine compounds is related to their base

content (piperazine-hexahydrate) while the acid residue has

but slight effect (2l). The action of piperazine is exerted on

metabolic processes of worm muscles giving rise to neuromuscular

block and reversible paralysis (21, 18, 20, 8, 11).

Different dosage schedules may be used in the treatment

of ascaridosis; administration for 4-5 days is generally

adopted for individual medication while for mass treatment a

single, large dose is given. By the mass treatment problems of

administration be a drawback to the efficacy of therapy. Thus

for a „Single dose" (3-4 gm of piperazine base) one needs to

take 8-10 or even more pills.

There are few contraindications to the use of piperazine; side

effects are rarely encountered and are of slight intensity.


Nevertheless, some adverse, toxic effects on the nervous system

were reported in the early period of piperazine therapy (10, 6,

3, 4). Although symptoms of intolerance (vertigo, myasthenia,

tremor, disorders of motility and coordination, epileptiform

convulsions) disappear soon after stopping the medication,piperazine

cannot he administered to patients with latent and manifest

epilepsy or to those with an increased spasmophilia (9).

The drug can he given only with great caution to patients with a

case history of allergy.Since piperazine does not destroy the

roundworms sensitisation due to released allergens will not

occur. However, allergic symptoms can occasionally he observed

during treatment.

In spite of the high efficacy of piperazine,these disadvantages

provide reasons for seeking new anthelminthics which may be

available as drugs of choice,for both individual and large scale

medication of ascaridosis.

Papers on the suitable anthelminthic action of a new drug, tetramisole

(Janssen), were first published in veterinary journals

and more recently in medical periodicals.

The active ingredient of tetramisole is represented by 2, 3, 5,

6-tetrahydro-6-phenyl-imidazo/2, 1-6/ thiazohydrochloride. It

is a white, stable, crystalline compound, soluble up to 21 fo in

water of 20° C,and is characterized by a melting point of 264° 0.

According to pharmacological studies carried out so far the new

drug is non-toxic and well tolerated. Its therapeutic index,

studied in a number of species,has proved favourable; the drug,

having exerted its effects,is eliminated from the host organism

within a few hours. Tetramisole and its metabolic products are

excreted in faeces, urine and bronchial mucus. Comprehensive

laboratory investigations revealed no change in the blood

picture or urine, nor in liver function resulting from use of

the drug (22).


The anthelminthic action of tetramisole is due to selective interference

with succinate-dehydrogenase activity of worm muscles

which leads to paralysis. The drug has a broad anti-nematode

spectrum being effective in animals against Haemonchus contortus

, Ostertagia circumcincta, Trichostrongylus spp., Nematodirus

spp., Chabertia ovina, Oesophagostomum Columbianum, Dictyocaulus

filaria and D. viviparus (12, 17, 13, 15, 14-, 16), also

against Ascaridia galli, Heterakis gallinarum, Capillaria obsignata

(5), as well as Hyostrongylus rubidus and Ascaris suum

(19). It has no effect on tapeworms, flukes, protozoa or bacteria.

A single, small dose of tetramisole has an effect of 68-

93 fo on Ascaris lumbricoides infection in human literature (2,7,

1, 23) without any marked undesirable and toxic effect. Reports

on its efficacy in hookworm infection are not in complete agreement.

A single dose of 2,5 mg/kg body weight generally exerted

an effect of 60 fo. In a study covering only a few cases it

proved ineffective in trichurosis and was only slightly effective

in enterobiosis (22).

Prior to clinical trials the study described in this paper was

designed to obtain some information on the in vitro action of

tetramisole and on the mechanism involved.

Materials and Methods

Ascaris suum was employed as the test worm on which to study

the anthelminthic effect of tetramisole in vitro. According to

many authors this species is identical with the nematode parasite

of man,Ascaris lumbricoides. Albeit there exist objections

to this opinion, all experts do agree that the biological and

epidemiological features of porcine Ascaris are closely related

to those of human Ascaris.

Worms obtained from pigs killed in a slaughterhouse were transported

to the laboratory in a vacuum flask containing physiological

saline solution at 37° C. The most vigorous specimens of


medium size adult worms were selected, washed repeatedly in

physiological saline, and placed in glass vessels. Male and

female worms were allocated in equal number in each series of

experiments. Physiological saline was used for control preparations

and for dissolving tetramisole;according to some workers,

and to our preliminary studies, worms can be kept alive in physiological

saline for 8-10 days.

Erlenmayer-flasks of 300 ml capacity,containing 200 ml of testsolution,

were used in the experiments. Dilutions of tetramiso­

le were prepared as follows:

1,00 mg/200 ml

0,50 mg/200 ml

0,10 mg/200 ml

0,05 mg/200 ml

0,01 mg/200 ml

5,00 /tg/ml,

2,50 /cg/ml,

0,50 //g/ml,

0,25 /teg/ml,

0,05 /fcg/ml.

Worms in test solutions were incubated at 37 C .

In the first series the effect of each dilution was examined

on 20 worms in each bath, and the procedure was repeated in two

other trials. Vigour was estimated on the spontaneous motion of

the worms and on that caused by mechanical as well as light irritation

(18). The readings were taken at 1, 2, 3, 4, 5, 6, 12,

24, 48, 72 and 96 hours.

After evaluation of the first experiment the effect was observed

also at 20, 40, 60, 80, 120 and 180 minutes to make the experiment

more complete; ten parallel test-worms were used for each

concentration, and the procedure was repeated once.

A study on the reactivation of worms exposed to the drug was

carried out in the second series of experiments. Worms, having

been exposed to the drug for 3 hours, were rinsed with and put

into physiological saline and incubated at 37° C; their behaviour

was observed at 20, 40, 60, 80, 120, 180 mi-


nutes and at 24, 48, 72 hours; to check vitality at 72 hours

thermo-irritation was also

carried out.

Results

The result of the first series of experiments is shown in

Talles 1 and 2. The worms were paralysed by low concentration

of tetramisole shortly after coming in contact with i t . At a

concentration of 1 mg tetramisole/200 ml more than 50 $ of the

worms stopped moving within 20 minutes, while all worms had

done so after 80 minutes. In the solution containing 0,5 mg

drug/200 ml paralysis of all worms was complete by 3 hours.

Although a paralysing effect at lower concentrations (0,10 and

0,05 mg/200 ml) was observed from the outset of the exposure,

i t was complete on all worms only by 48 and 72 hours,respectively.

The effect of 0,01 mg drug/200 ml was first detected after

an exposure of 48 hours and i t caused paralysis in about one

half of the worms by 96 hours.No sex differences in sensitivity

to the drug were found. According to readings at 24, 48, 72 and

96 hours the worms failed to reactivate in tetramisole.

Observations carried out at higher concentrations at 20 minute

intervals revealed that cessation of worm movement was not

preceded by an excitation phase.

The results of the second series of experiments are summarized

in Table 3- I t can be seen that the paralysing effect of tetramisole

is a reversible one; the worms recover in normal saline.

The time required for reactivation is dependent on the drug

concentration to which the worms had been previously exposed;

the higher the concentration that had been used the longer was

the period needed for recovery.


Paralysing Effect of Tetramisole (tested on 60-60 worms)

A különböző Tetramisole-koncentrációk paralysáló hatásának időbeli

kialakulása órákban (60-60 teszt-féreg in vitro vizsgálata alapján)

Time

(hours)

leolvasás

ideje

( óra)

Tetramisole concentration (mg in 200 ml)

Tetramisole-koncentráció 200 ml-ben Control

Kontroll

o 0 ,5 0 1 0, 05 0,01

+ - + - + - + - + - + -

1 3 57 33 27 41 19 60 - 60 - 20 -

2 1 59 4 56 38 22 42 18 60

-

20 -

3 - 60 - 60 32 28 42 18 60 - 20 -

4 - 60

-

60 31 29 40 20 60 - 20 -

5 - 60 - 60 25 35 38 22 60

-

20 -

6 - 60 - 60 25 35 32 28 60 - 20 -

12 - 60 - 60 15 45 31 29 60

-

20 -

24 - 60 - 60 9 51 19 41 60 - 20 -

48

-

60 - 60

-

60 3 57 51 9 20 -

72

-

60

-

60 - 60 - 60 42 18 20

-

96

-

60 - 60 60

-

60 36 24 20 -


Paralysing Effect of Tetramisole (tested on 20-20 worms)

A különböző Tetramisole-koncentrációk paralysáló hatásának időbeli

kialakulása percekben (20-20 teszt-féreg in vitro vizsgálata alapján)

Time

(minutes)

Leolvasás

ideje

(perc)

Tetramisole concentration (mg in 200 ml)

Tetramisole koncentráció 200 ml-hen Control

Kontroll

1 0,5 1 0,05 0,01

+ - +

-

+ - + - + - + -

20 8 12 17 3 19 1 20 - 20 - 20 -

40 3 17 12 8 17 3 20 20 - 20

-

60 1 19 11 9 15 5 20 - 20 - 20 -

80 - 20 5 15 15 5 19 1 20

-

20

-

120 - 20

-

20 13 7 16 4 20 - 20 -

180 - 20

-

20 9 11 15 5 20 - 20

-

+ = motile roundworms - motilis orsóféreg

- = paralysed roundworms - bénult orsóféreg


Table 3-3.

táblázat

Reactivation of Roundworms, Exposed to Tetramisole for 3 Hours,

an Physiological Saline Solution (tested on 20-20 worms)

Az orsóférgek reaktiválódása fiziológiás sóoldatban, háromórás

Tetramisole előkezelés után (20-20 teszt-féreg vizsgálata alapján)

Time

Leolvasás

ideje

(perc)

(óra)

Concentration of Tetramisole (mg/200 ml)

Előkezelésnél alkalmazott Tetramisole-koncentráció Control

Kontroll

1 0 ,5 o 1 0,05 0,01

+

- + - + - + - + - + -

20 minutes _ 20 - 20 2 18 4 16 20 - 20 —

4-0 minutes 2 18

-

20 4 16 10 10 20

-

20

-

60 minutes 1 19 2 18 8 12 11 9 20 - 20 -

80 minutes - 20 3 17 8 12 12 8 20

-

20 -

120 minutes - 20 - 20 12 8 12 8 20

-

20 -

180 minutes 2 18 3 17 15 5 11 9 20

-

20 -

24 hours 6 14 18 2 20

- 19 1 20 - 20 -

48 hours 8 12 19 I 20

-

20 - 20 - 20

-

72 hours 18 2 19 1 20 - 20 - 20

-

20

-


Discussion

In vitro studies on both the efficiency of tetramisole and the

mechanism involved have shown that a paralysing effect was

exerted on Ascaris suum in low concentrations of the drug. The

effect was produced in a short time. Because of the lack of

excitation phase prior to paralysis this drug meets one of the

basic requirements for safe and efficient therapy of ascaridosis.

The paralysing effect of tetramisole proved reversible. The

curve of regeneration is nearly symmetrical with that of

action's development. Irreversible damage of worms may occasionally

result from higher concentrations of the drug.

Acknowledgements

Our thanks are due to the Medical Department of Kőbányai Pharmaceutical

Company for the supply of tetramisole.

LENGYEL, A. — ZOLTAI, L.: A tetramizol hatásának in vitro

vizsgálata Ascaris

suumra

A szerzők in vitro kisérletsorozataikban különböző tetramizolkoncentrációk

(l, 0,5, 0,1, 0,05 és 0,01 mg/200 ml) paralysaló

hatásának időbeli kialakulását vizsgálták, mindkét nembeli, közepes

fejlettségű Ascaris suum teszt-férgeken. Legkifejezettebb

hatást az 1 mg és 0,5 mg/200 ml koncentrációk mutattak, amelyek

80 perc, illetve 3 órás expozición belül valamennyi teszt-féreg

bénulását előidézték. A paralysis kifejlődését excitatiós szakasz

nem előzte meg. A bénulás reverzibilis, élettani konyhasóoldatban

a férgek visszanyerték motilitásukat. A reaktiválódás

ideje az előzetes hatóanyag-koncentráció függvénye.


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Received: 25-4.1970.

Dr. A. LENGYEL,

Dr. L. ZOLTAI,

Department of Parasitology National

Institute of Public Health,

Budapest, IX., Gyáli ut 2-6.

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