Journal ofHelmintholoxy (1976) 50. 21—28
Studies with Brugia pahangi 12. The activity of levamisole ROSEMARY ROGERS and D. A. DENHAM Department of Medical Helminthology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT
ABSTRACT The effects of levamisole on adults, third stage infective larvae, and microfilariae of Brugia pahangi were studied in in vitro culture and in vivo against developing stages in the vector mosquito and in infected cats. In vitro the drug was effective only at dose levels much higher than can be tolerated by mammals. It was active against the developmental stages of the worm in the vector Aedes aegypti. The drug was strongly microfilaricidal in cats but less effective against adult worms.
Tetramisole is a potent broad-spectrum anthelmintic whose active principle is the levorotatory isomer levamisole (Thienpont ct al., 1966). It probably acts by inhibiting the succinic dehydrogenase pathway (Van den Bossche, 1972; Van den Bossche and Janssen, 1969). Pharmacological aspects of tetramisole were discussed by Van Neuten (1972). The mode of action of levamisole on Ascaris was discussed by Van den Bossche and Janssen (1967) and on other intestinal helminths by Gras (1974). Coles and Jenkins (personal communication) showed in vitro paralysis of adult Nippostrongylus brasiliensis by levamisole but found that, if the worms were left in the drug, they recovered their activity. We have previously studied the effects of tetramisole (the D/L isomer mixture) on microfilariai levels and on adult worms in cats infected with Bntgia pahangi (unpublished). The present study was undertaken to observe the effect of levamisole on B. pahangi in vitro and in vivo, in vector mosquitoes and in cats. MATERIALS AND METHODS
.
(1) In vitro studies Adult male and female Brugia pahangi dissected from the lymphatics of infected cats (Denham ct al., 1971) were washed in Medium 199 (Wellcome), containing 100 units/ml Streptomycin sulphate (Glaxo), 500 units/ml Penbritin (Beecham) and 100 units/ml Mycostatin (Squibb). For control cultures, worms were transferred individually on the point of a needle to 12 x 75 mm sterile, clear, polythene tubes (Falcon, No. 2058), containing 1 ml of the above medium plus 10% inactivated horse serum (Wellcome, No. 5). The tubes were sealed with tightly-fitting caps and maintained at 37°C. For experimental cultures, worms were placed individually in various concentrations of levamisole, made by diluting a 1 % solution of levamisole hydrochloride in the above culture medium and maintained at 37°C. Cultures were observed at 5 mins, 15 mins, 30 mins and then at half-hourly or hourly intervals, using a Zeiss Inverted microscope. Third stage infective larvae were obtained by crushing mosquitoes (Aedes aegypti, f strain) infected for 10 days and collecting the larvae by means of a Baermann apparatus w
21
R. ROGERS and D. A. DENHAM
filled with Medium 199. The larvae were washed several times in Medium 199 plus antibiotics. Five to 10 worms were placed in each tube, containing culture medium only, or various concentrations of levamisole. The cultures were maintained and observed in the same way as those of adult worms. One ml of blood from a cat with a high microfilarial level was passed through a 5 y.m mesh Nucleporefilterheld in a Millipore(Swinnex-25)filterholder and thefilterwas washed with Medium 199. Thefilterwas placed in a tube containing culture medium and agitated to dislodge microfilariae from the filter. A drop of the concentrated microfilarial suspension was placed in each well of a microtitre tray (Cooke Microtiter System) and 0-2 ml of culture medium or various concentrations of levamisole solution were added. The tray was covered with a glass plate to prevent evaporation and maintained at 37°C. The cultures were observed with the Zeiss Inverted microscope. (2) Studies with infected Aedes aegypti A.aegypti (P" strain) females were fed on an anaesthetised cat infected with B. pahangi, with a microfilarial level of 30-50 microfilariae/20 mm3 blood. Thirty engorged females were aspirated into each of a series of small cardboard cartons and fed on various concentrations of levamisole in a 10% sucrose solution or upon 10% sucrose solution without drug. Mosquitoes exposed to drug solutions were not given any other liquid so that they had to drink the test solution. The mosquitoes were dissected 10 days later and the numbers and condition of larvae from each mosquito were noted. (3) In vivo studies in cats infected with B. pahangi Four cats with patent B. pahangi infections were treated with levamisole, three at 25 mg/kg/day for five consecutive days, intraperitoneally (the fourth died one hour after the first treatment). Microfilarial levels were followed before, during, and after treatment. One of the cats was autopsied 20 days after the first treatment, according to the method described by Denham et ah (1971). The second cat had a biopsy of the infected left hind leg lymphatic taken 39 days after treatment and was autopsied 18 days later, i.e. 57 days after treatment. The third cat had a lymphatic biopsy taken 15 days after treatment and was killed the following day. RESULTS (1) Effect of levamisole on adult B. pahangi in vitro In control cultures male and female worms showed active motility throughout the 24 hours of observation. These worms, together with the survivors of levamisole treatment after 24 hours, were kept in culture, the medium being changed daily. Seven females and two males survived for 14 days; six of these females lived for 38 days and one female survived for 61 days. Worms in cultures with levamisole concentrations below 0015%, coiled and moved actively during the 24 hours of observation and survived after this. Both sexes, in 0-02% solutions, showed reduced motility in the first hour, but were then very active up to 24 hours later. In 0-03 and 0-04% solutions, the worms showed hyperactive coiling at 2-6 hours and 5-6 hours respectively, but were otherwise normally active for 24 hours. A male worm treated at 0-05 % became completely immobile at 5 hours, but regained slight motility at 17 hours. In 0*1 % solutions, female worms were very active for 24 hours, showing peak motility at 5-6 hours. The males, however, were totally immobilized in 1 hour. Concentrations of 0*5 and 1*0% inactivated the worms completely in 5 mins and they did not regain motility when washed in fresh medium after 18 hours. 22
:/
(2) Effect of levamisole on third stage infective larvae in vitro
Studies with Brugia pahangi 12
•,••
In control cultures, worms were motile throughout the 100 hours of observation and in some cultures survived 16-17 days, with a daily change of medium after the 100-hour period of test observation. (Third stage worms from the peritoneal cavity of Meriones unguicuiatus, placed in culture after 3-6 days in the host, moulted at 5-8 days.old in culture and survived for up to 21 days.) Table 1 shows the time and concentration of levamisole needed to immobilize all worms in culture. At levels below 0-04% the worms retained mobility up to 100 hours. The LD50 for the drug at 24 hours was 0-04%. TABLE 1 Levamisole concentrations and time required to kill all B. pahangi third stage infective larvae in vitro Levamisole concentration (%) 1*0 0-5 0-1,009 008,0075 007, 006, 005 004
Time (hours) 5mins 30 mins 1 H 18 50-60
- (3) Effect of levamisole on B. pahangi microfilariae in vitro Table 2 shows the concentrations of levamisole and the time necessary to kill all microfilariae in a culture. TABLE 2 oncentration and time required to kill all B. pahangi Levamisole concentration (%) 1-0-0-5 04—0-2 01 009 008 007 006 005 004
003—001
Time (hours) 15 mins
21
3—3i 3J-4
41
6 7—10 12—22 24 24
(4) Effect of levamisole on B. pahangi in the vector A. aegypti Concentrations of 1*0 and 0*1 % levamisole killed all the mosquitoes by days four and seven respectively. These were dissected immediately. One mosquito fed on the 1 % solution contained five 2nd (sausage-)' stage larvae and the rest were uninfected. Mosquitoes fed on 23
R. ROGERS and D. A. DENHAM
0*1 % levamisole contained a few sausage-stage larvae and very small third stage larvae (see Table 3). Table 3 shows, for each drug concentration, the number of mosquitoes infected, the average number of larvae recovered per dissected mosquito and the average per infected mosquito. With all concentrations down to 0-008 %, stunted third stage or sausage-stage larvae were recovered. 0-001 % levamisole had no effect on the worms and the numbers recovered were comparable with those from control mosquitoes. TABLE 3
-
Effect of levamisole on B. pahangi in the vector A. aegyptl (s = second, sausage-stage, larvae). Levamisole , concentration (%)
Number of mosquitoes infected
Average larvae per dissected mosquito
Average larvae per infected mosquito
1-0
1/22 (all dead day 4)
0-22s
5-OOs
0-1
7/16 (all dead day 7)
1-44 0-69s
l-75s l-57s
008
20/24
0-50 l-75s
0-50 2-10s
005
14/21
1-05 0-66s
1-57 1-OOs
003
17/23
0-88
0-64 •
l-56s
*-•••'••' 2 « 1 2 s
001
14/22
1-41 0-18s
2-21 0-29s
001
24/30
2-77 2-67s
0-13 3-34s
24/25
4-16 0-16s
0008 i
'
•
:
4-33 0-17S
0001
22/24
6-86
7-50
Control 1
28/30
4-37
5-68
Control 2
14/14
6-84
6-85
Control 3
28/30
8-42
905
(5) Effect of levamisole on microfilariae and adult B. pahangi in cats In preliminary experiments we found that tetramisole (a mixture of the D and L isomers) had no effect on B. pahangi in cats. Used at levels of 15, 20 and 25 mg/kg/day, intramuscularly forfivedays, no decrease in microfilarial levels was noted and adult worms showed no abnormality when recovered from the lymphatics at autopsy. The highest dose of drug caused severe distress to the animals for several hours after treatment. The results obtained in the present study are as follows: Cat M 44 was given levamisole at 25 mg/kg/day for five days intraperitoneally. After each treatment the cat was distressed and salivated for several hours. The microfilarial 24
' - Studies with Brugia pahangi 12
count prior to treatment was 107 mf/20 mm3 blood. This count dropped to zero within an hour of the first treatment and remained zero until the cat was autopsied 20 days later. All 17 male and 42 female worms recovered from the hind limb lymphatics were normal and active. The females were large and contained fully-developed microfilariae. Cat M 42 was similarly treated and showed similar distress soon after each treatment. The initial microfilarial count of 52 mf/100 mm3 blood dropped to 3/100 in \ hour and 0/100 by \\ hours after the first treatment. The following day, 3 mf/100 mm3 were found before treatment, but the count was again zero 2\ hours later and remained so until 15 days after the first treatment, when 1/100 mm3 was counted. For the next few weeks, the microfilarial count rose slowly to 2, 3, 4, 7 mf/100 mm3 blood and at autopsy, 57 days after treatment, the count was 8/100 mm3. The afferent lymphatic biopsy taken on day 39, showed a much enlarged, fibrous lymphatic, with pockets of dead, broken worms (6-8) in lymphoid tissue almost occluding the vessel. A new, fine lymphatic had developed in the leg. One live, female worm, with microfilariae in utero, was recovered. At autopsy, the left leg had healed after the operation. Two fine afferent lymphatics had developed to replace the biopsied tissue and there were two well-developed skin lymphatics. One female worm (containing abnormal embryos) and four dead worms were found in the old part of the lymphatic. Seven dead worms and one female with abnormal embryos were recovered from the right afferent lymphatic and four dead worms were found in the afferent sinus of the popliteal lymph node. Cat A5 was severely distressed, salivated and was unable to eat during the period of treatment, but had recovered the following week. The microfilarial count before treatment ..was 26/20 mm3 blood. Half an hour after treatment the count was 1/20 mm3. The following day it was zero and remained so until the cat was autopsied 16 days after thefirsttreatment. A biopsy of the infected left hind leg lymphatic yielded two dead, amorphous worms. At autopsy, a left hind leg skin lymphatic contained about 10 dead worms which were white calcified masses embedded in lymphoid tissue. Another 12 dead worms were almost whole but coated in lymphoid tissue and one was in an haemorrhagic area. In the afferent popliteal lymphatic there were about 15 dead, degenerating worms in lymphoid tissue and another eight surrounded by haemorrhage. From the infected right hind leg, a skin lymphatic yielded one dead worm in haemorrhage and a by-pass vessel contained one dead worm in lymphoid tissue. Cat A16 had a microfilarial count of 148/10 mm3 before treatment, which became 0/20 mm3 half an hour after treatment. One hour after treatment the cat died, with blood and saliva pouring from mouth and nose. The lungs were haemorrhagic. DISCUSSION Levamisole showed activity in vitro against the adult worms, infective larvae and microfilariae of B. pahangi, but the dose levels needed to immobilize the worms were very high, compared with the level used in vivo. The infected cats were treated with levamisole at 125 mg/kg. In vitro, however, to immobilize adult worms, infective larvae and microfilariae respectively in 5 mins, 5000, 10000 and 5-10000 mg/kg was the equivalent dosage needed. The LD50 at 24 hours for infective larvae was equivalent to 400 mg/kg and microfilariac were immobilized at 24 hours by the same level of drug. Natarajan et ah (1974) found that all three stages of Breinlia sergenti were susceptible to the drug. The infective larvae and microfilariae became sluggish after 15 mins in a 0-1 % solution, while adult female worms lost motility on contact with a 001 % solution. 25
R. ROGERS and D. A. DENHAM
The increased muscular tone observed in their adultworms with high drug concentrations, was seen with B. pahangi adults, as shown by the hyperactive coiling noted above. Zaman and Natarajan (1973) found that adult Br. sergenti in the slow loris were killed by much higher doses of levamisole than those needed to kill microfilariae (4 x 50 mg/kg killed adults and microfilariae, while doses, on alternate days, of 1-32 mg/kg killed all microfilariae). The same amounts of diethylcarbamazine (DEC) were only partially active against either stage of the worm. Adult B. pahangi in cats are killed by DEC at 50 mg/kg for five days. The LD50 for B. pahangi infective larvae at 24 hours in vitro is 0-2% DEC, compared with 0-04% levamisole (unpublished data). More than 0-2% DEC is required to kill all B. pahangi microfilariae in 24 hours in vitro, whilst 0-04 % levamisole is adequate. 0-3 % DEC and 0-07 % levamisole kill microfilariae in 6 hours. Levamisole is thus effective against all three stages of the parasite at much lower levels than DEC in vitro but the levels are much higher than those which can be used in vivo. The reversible paralysis which Coles and Jenkins (unpublished) noted when adult N. brasiliensis were exposed to 0-01 % levamisole in vitro did not occur with B. pahangi. They postulated that a neuro-receptor in the worm was continually stimulated by the high dose of drug, causing paralysis, but that later the receptor could no longer be stimulated and the worms became motile again. Levamisole was a fairly effective filaricide against the developmental stages of B. pahangi in mosquitoes, even at quite low levels (0-008%). Gerberg et.al. (1972) found that levamisole had a detrimental effect on the development of Dirofilaria immitis in A. aegypti. Although levamisole has some filaricidal activity in vitro and in mosquitoes, it had a limited effect on B. pahangi in cats. Levamisole (but not tetramisole) had an immediate microfilaricidal effect, but some adult female must still have produced microfilariae long after treatment, as the microfilarial level of M42 rose again after initially being reduced to zero. The adult worms from M44 were all alive and undamaged. Some worms from M42 were dead and some females contained abnormal embryos, indicating that perhaps the drug had a much longer-term effect on the worms than could be detected in the cat killed 20 days after treatment (although some worms may have been dead before drug treatment). Mak et ah (1974) found that levamisole hydrochloride in total doses of 30-150 mg/kg body weight had micro- and macrofilaricidal effects on subperiodic B. malayi infections in cats. Zaman and Lai (1973) treated two patients infected with Wuchereria bancrofti with levamisole. A single dose of 120 mg caused a marked reduction in the numbers of circulating microfilariae, but several doses were needed to reduce the count to zero. One patient, after one treatment, developed side effects similar to those occurring after DEC treatment. They concluded that this reaction was due to destruction of the parasites and that caution would be needed in treating patients with high microfilaraemia. Zaman and Fung (1973) successfully treated a case of eosinophilic lung with levamisole. O'Holohan and Zaman (1974) found levamisole to be microfilaricidal in humans infected with B. malayi, but adult worms were not killed or sterilized by the dosages used. Tulloch and Anderson (1972) found levamisole to be prophylactic against D. immitis infections in dogs. Duke (1974) found that levamisole made chimpanzees infected with Onchocerca volvulus ill, at doses usually used for intestinal nematodes. However, when injected intra26
Studies with Brugia pahangi 12
muscularly at 10 mg/kg for 15 days (a total dose of 150 mg/kg or 2-4 g), microfilarial counts became very low and remained at zero after complete clearance of microfilariae with DEC. Thus microfilariae were affected by levamisole and the adult worms were probably killed or sterilized. (The animals were not necropsied, so that the state of the adult worms could not be determined.) Duke (1975) treated O. volvulus in a chimpanzee and a human, giving the drug intramuscularly at 2 and 2-5 mg/kg for up to 15 days. The maximum dose tolerated was 2 mg/kg. No great effect was observed on either microfilariae or adult worms. The main conclusions which can be drawn from the present work are thus: (1) that the in vitro action of levamisole on B. pahangi occurred with higher concentrations than against Br. sergenti and than can be tolerated in vivo. (2) that levamisole is active against the developmental stages of B. pahangi in the mosquito vector. (3) that levamisole is very active against microfilariae in vivo, but less active against adult worms. It appears to be much more active on Br. sergenti in vivo than against B. pahangi and O. volvulus. In view of the above observations and the relatively high toxicity of the drug (shown by the side effects after treatment of cats and the death of one), it would seem that there will be no great potential for levamisole for use in human filariasis. REFERENCES DENHAM, D. A., PONNUDURAI, T., NELSON, G. S., GUY, FRANCES and ROGERS, ROSEMARY (1971) The effect of Metrifonate on Brugia paliangi infections in domestic cats. Bulletin of the World Health Organization, 45, 423-429. DUKE, B. O. L. (1974) The effect of Levamisole on Onchocerca volvulus. Transactions of the Royal Society of Tropical Medicine and Hygiene, 68, 71. DUKE, B. O. L. (1975) Further trial of Levamisole against Onchocerca volvulus. Transactions of the Royal Society of Tropical Medicine and Hygiene, 69, 287. GERBERG, E. J., KUTZ, F. W. and TULLOCH, G. S. (1972) Preliminary evaluation of 1-Tetramisole as an antifilarial in a mosquito test system. In Canine Heartworm Disease. The current knowledge (Ed. R. E. Bradley). Florida, pp 105-108. GRAS, G. (1974) Les anthelminthiques intestinaux; aspects pharmacologiques. Medecine d'Afriqtte Noire, 21,11-28. MAK, J. W., ZAMAN, V. and SIVANANDAM, S. (1974) Antifilarial activity of Levamisole hydrochloride against subperiodic Brugia malayl infection of domestic cats. American Journal of Tropical Medicine and Hygiene, 23, 369-374. NATARAJAN, P. N., ZAMAN, V. and YEOH, T. S. (1974) In vitro activity of Levamisole on the infective larvae, microfilariae and adult worms of Breinlia sergenti. InternationalJournal for Parasitology, 4,207-210. O'HOLOHAN, D. R. and ZAMAN, V. (1974) Treatment of Brugia malayl infection with Levamisole. Journal of Tropical Medicine and Hygiene, T7t 113-115. THIENPONT, D., VANPARIJS, O F. J., RAEYMAEKERS, A. H. M., VANDEBERK, J., DEMOEN, P. J. A., ALLEWIJN, F. T. N., MARSBOOM, R. P. H., NIEMEGEERS, C. J. E.. SCHELLEKENS, K. H. L. and JANSSEN, P. A. (1966) Tetramisole (R8299)—a new potent broad spectrum anthelmintic. Nature (London), 209,1084-1086. TULLOCH, G. S. and ANDERSON, R. A. (1972) Tetramisole and canine dirofilariasis. In Canine Heartworm Disease. Tfte current knowledge. Ed R. E. Bradley). Florida, pp 101-104. VAN den BOSSCHE, H. (1972) Biochemical effects of Tetramisole. In Comparative Biocliemistry ofParasites (Ed H. Van den Bossche). Academic Press: New York, pp 117-125. VAN den BOSSCHE, H. and JANSSEN, P. A. (1967) Mode of action of Levamisole on Ascaris. Life Sciences, 6, 1781-1792. VAN den BOSSCHE, H. and JANSSEN, P. A. J. (1969) The biochemical mechanism of action of the antincmatodal drug Tetramisole. Biochemical Pharmacology, 18,35-42. 27
R. ROGERS and D. A. DENHAM VAN NEUTEN, J. M. (1972) Pharmacological aspects of Tetramisole. In Comparative Biochemistry of Parasites (Ed H. Van den Bossche). Academic Press: New York, pp 101-115. ZAMAN, V. and FUNG, W. P. (1973) Treatment of eosinophilic lung with Levamisole. Transactions of the Royal Society of Tropical Medicine and Hygiene, 67, 144-145. ZAMAN, V. and LAL, M. (1973) Treatment of Wuchereria bancrofti with Levamisole. Transactions of the Royal Society of Tropical Medicine and Hygiene, 67, 610. ZAMAN, V. and NATARAJAN, P. N. (1973) The antifilarial activity of Levamisole on Breinlia sergenti compared with diethylcarbamazine. Journal of Tropical Medicine and Hygiene, 76, 126-128. Accepted 2. October 1975.
28
Studies with Brugia pahangi 12. The activity of levamisole ROSEMARY ROGERS and D. A. DENHAM Department of Medical Helminthology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT
ABSTRACT The effects of levamisole on adults, third stage infective larvae, and microfilariae of Brugia pahangi were studied in in vitro culture and in vivo against developing stages in the vector mosquito and in infected cats. In vitro the drug was effective only at dose levels much higher than can be tolerated by mammals. It was active against the developmental stages of the worm in the vector Aedes aegypti. The drug was strongly microfilaricidal in cats but less effective against adult worms.
Tetramisole is a potent broad-spectrum anthelmintic whose active principle is the levorotatory isomer levamisole (Thienpont ct al., 1966). It probably acts by inhibiting the succinic dehydrogenase pathway (Van den Bossche, 1972; Van den Bossche and Janssen, 1969). Pharmacological aspects of tetramisole were discussed by Van Neuten (1972). The mode of action of levamisole on Ascaris was discussed by Van den Bossche and Janssen (1967) and on other intestinal helminths by Gras (1974). Coles and Jenkins (personal communication) showed in vitro paralysis of adult Nippostrongylus brasiliensis by levamisole but found that, if the worms were left in the drug, they recovered their activity. We have previously studied the effects of tetramisole (the D/L isomer mixture) on microfilariai levels and on adult worms in cats infected with Bntgia pahangi (unpublished). The present study was undertaken to observe the effect of levamisole on B. pahangi in vitro and in vivo, in vector mosquitoes and in cats. MATERIALS AND METHODS
.
(1) In vitro studies Adult male and female Brugia pahangi dissected from the lymphatics of infected cats (Denham ct al., 1971) were washed in Medium 199 (Wellcome), containing 100 units/ml Streptomycin sulphate (Glaxo), 500 units/ml Penbritin (Beecham) and 100 units/ml Mycostatin (Squibb). For control cultures, worms were transferred individually on the point of a needle to 12 x 75 mm sterile, clear, polythene tubes (Falcon, No. 2058), containing 1 ml of the above medium plus 10% inactivated horse serum (Wellcome, No. 5). The tubes were sealed with tightly-fitting caps and maintained at 37°C. For experimental cultures, worms were placed individually in various concentrations of levamisole, made by diluting a 1 % solution of levamisole hydrochloride in the above culture medium and maintained at 37°C. Cultures were observed at 5 mins, 15 mins, 30 mins and then at half-hourly or hourly intervals, using a Zeiss Inverted microscope. Third stage infective larvae were obtained by crushing mosquitoes (Aedes aegypti, f strain) infected for 10 days and collecting the larvae by means of a Baermann apparatus w
21
R. ROGERS and D. A. DENHAM
filled with Medium 199. The larvae were washed several times in Medium 199 plus antibiotics. Five to 10 worms were placed in each tube, containing culture medium only, or various concentrations of levamisole. The cultures were maintained and observed in the same way as those of adult worms. One ml of blood from a cat with a high microfilarial level was passed through a 5 y.m mesh Nucleporefilterheld in a Millipore(Swinnex-25)filterholder and thefilterwas washed with Medium 199. Thefilterwas placed in a tube containing culture medium and agitated to dislodge microfilariae from the filter. A drop of the concentrated microfilarial suspension was placed in each well of a microtitre tray (Cooke Microtiter System) and 0-2 ml of culture medium or various concentrations of levamisole solution were added. The tray was covered with a glass plate to prevent evaporation and maintained at 37°C. The cultures were observed with the Zeiss Inverted microscope. (2) Studies with infected Aedes aegypti A.aegypti (P" strain) females were fed on an anaesthetised cat infected with B. pahangi, with a microfilarial level of 30-50 microfilariae/20 mm3 blood. Thirty engorged females were aspirated into each of a series of small cardboard cartons and fed on various concentrations of levamisole in a 10% sucrose solution or upon 10% sucrose solution without drug. Mosquitoes exposed to drug solutions were not given any other liquid so that they had to drink the test solution. The mosquitoes were dissected 10 days later and the numbers and condition of larvae from each mosquito were noted. (3) In vivo studies in cats infected with B. pahangi Four cats with patent B. pahangi infections were treated with levamisole, three at 25 mg/kg/day for five consecutive days, intraperitoneally (the fourth died one hour after the first treatment). Microfilarial levels were followed before, during, and after treatment. One of the cats was autopsied 20 days after the first treatment, according to the method described by Denham et ah (1971). The second cat had a biopsy of the infected left hind leg lymphatic taken 39 days after treatment and was autopsied 18 days later, i.e. 57 days after treatment. The third cat had a lymphatic biopsy taken 15 days after treatment and was killed the following day. RESULTS (1) Effect of levamisole on adult B. pahangi in vitro In control cultures male and female worms showed active motility throughout the 24 hours of observation. These worms, together with the survivors of levamisole treatment after 24 hours, were kept in culture, the medium being changed daily. Seven females and two males survived for 14 days; six of these females lived for 38 days and one female survived for 61 days. Worms in cultures with levamisole concentrations below 0015%, coiled and moved actively during the 24 hours of observation and survived after this. Both sexes, in 0-02% solutions, showed reduced motility in the first hour, but were then very active up to 24 hours later. In 0-03 and 0-04% solutions, the worms showed hyperactive coiling at 2-6 hours and 5-6 hours respectively, but were otherwise normally active for 24 hours. A male worm treated at 0-05 % became completely immobile at 5 hours, but regained slight motility at 17 hours. In 0*1 % solutions, female worms were very active for 24 hours, showing peak motility at 5-6 hours. The males, however, were totally immobilized in 1 hour. Concentrations of 0*5 and 1*0% inactivated the worms completely in 5 mins and they did not regain motility when washed in fresh medium after 18 hours. 22
:/
(2) Effect of levamisole on third stage infective larvae in vitro
Studies with Brugia pahangi 12
•,••
In control cultures, worms were motile throughout the 100 hours of observation and in some cultures survived 16-17 days, with a daily change of medium after the 100-hour period of test observation. (Third stage worms from the peritoneal cavity of Meriones unguicuiatus, placed in culture after 3-6 days in the host, moulted at 5-8 days.old in culture and survived for up to 21 days.) Table 1 shows the time and concentration of levamisole needed to immobilize all worms in culture. At levels below 0-04% the worms retained mobility up to 100 hours. The LD50 for the drug at 24 hours was 0-04%. TABLE 1 Levamisole concentrations and time required to kill all B. pahangi third stage infective larvae in vitro Levamisole concentration (%) 1*0 0-5 0-1,009 008,0075 007, 006, 005 004
Time (hours) 5mins 30 mins 1 H 18 50-60
- (3) Effect of levamisole on B. pahangi microfilariae in vitro Table 2 shows the concentrations of levamisole and the time necessary to kill all microfilariae in a culture. TABLE 2 oncentration and time required to kill all B. pahangi Levamisole concentration (%) 1-0-0-5 04—0-2 01 009 008 007 006 005 004
003—001
Time (hours) 15 mins
21
3—3i 3J-4
41
6 7—10 12—22 24 24
(4) Effect of levamisole on B. pahangi in the vector A. aegypti Concentrations of 1*0 and 0*1 % levamisole killed all the mosquitoes by days four and seven respectively. These were dissected immediately. One mosquito fed on the 1 % solution contained five 2nd (sausage-)' stage larvae and the rest were uninfected. Mosquitoes fed on 23
R. ROGERS and D. A. DENHAM
0*1 % levamisole contained a few sausage-stage larvae and very small third stage larvae (see Table 3). Table 3 shows, for each drug concentration, the number of mosquitoes infected, the average number of larvae recovered per dissected mosquito and the average per infected mosquito. With all concentrations down to 0-008 %, stunted third stage or sausage-stage larvae were recovered. 0-001 % levamisole had no effect on the worms and the numbers recovered were comparable with those from control mosquitoes. TABLE 3
-
Effect of levamisole on B. pahangi in the vector A. aegyptl (s = second, sausage-stage, larvae). Levamisole , concentration (%)
Number of mosquitoes infected
Average larvae per dissected mosquito
Average larvae per infected mosquito
1-0
1/22 (all dead day 4)
0-22s
5-OOs
0-1
7/16 (all dead day 7)
1-44 0-69s
l-75s l-57s
008
20/24
0-50 l-75s
0-50 2-10s
005
14/21
1-05 0-66s
1-57 1-OOs
003
17/23
0-88
0-64 •
l-56s
*-•••'••' 2 « 1 2 s
001
14/22
1-41 0-18s
2-21 0-29s
001
24/30
2-77 2-67s
0-13 3-34s
24/25
4-16 0-16s
0008 i
'
•
:
4-33 0-17S
0001
22/24
6-86
7-50
Control 1
28/30
4-37
5-68
Control 2
14/14
6-84
6-85
Control 3
28/30
8-42
905
(5) Effect of levamisole on microfilariae and adult B. pahangi in cats In preliminary experiments we found that tetramisole (a mixture of the D and L isomers) had no effect on B. pahangi in cats. Used at levels of 15, 20 and 25 mg/kg/day, intramuscularly forfivedays, no decrease in microfilarial levels was noted and adult worms showed no abnormality when recovered from the lymphatics at autopsy. The highest dose of drug caused severe distress to the animals for several hours after treatment. The results obtained in the present study are as follows: Cat M 44 was given levamisole at 25 mg/kg/day for five days intraperitoneally. After each treatment the cat was distressed and salivated for several hours. The microfilarial 24
' - Studies with Brugia pahangi 12
count prior to treatment was 107 mf/20 mm3 blood. This count dropped to zero within an hour of the first treatment and remained zero until the cat was autopsied 20 days later. All 17 male and 42 female worms recovered from the hind limb lymphatics were normal and active. The females were large and contained fully-developed microfilariae. Cat M 42 was similarly treated and showed similar distress soon after each treatment. The initial microfilarial count of 52 mf/100 mm3 blood dropped to 3/100 in \ hour and 0/100 by \\ hours after the first treatment. The following day, 3 mf/100 mm3 were found before treatment, but the count was again zero 2\ hours later and remained so until 15 days after the first treatment, when 1/100 mm3 was counted. For the next few weeks, the microfilarial count rose slowly to 2, 3, 4, 7 mf/100 mm3 blood and at autopsy, 57 days after treatment, the count was 8/100 mm3. The afferent lymphatic biopsy taken on day 39, showed a much enlarged, fibrous lymphatic, with pockets of dead, broken worms (6-8) in lymphoid tissue almost occluding the vessel. A new, fine lymphatic had developed in the leg. One live, female worm, with microfilariae in utero, was recovered. At autopsy, the left leg had healed after the operation. Two fine afferent lymphatics had developed to replace the biopsied tissue and there were two well-developed skin lymphatics. One female worm (containing abnormal embryos) and four dead worms were found in the old part of the lymphatic. Seven dead worms and one female with abnormal embryos were recovered from the right afferent lymphatic and four dead worms were found in the afferent sinus of the popliteal lymph node. Cat A5 was severely distressed, salivated and was unable to eat during the period of treatment, but had recovered the following week. The microfilarial count before treatment ..was 26/20 mm3 blood. Half an hour after treatment the count was 1/20 mm3. The following day it was zero and remained so until the cat was autopsied 16 days after thefirsttreatment. A biopsy of the infected left hind leg lymphatic yielded two dead, amorphous worms. At autopsy, a left hind leg skin lymphatic contained about 10 dead worms which were white calcified masses embedded in lymphoid tissue. Another 12 dead worms were almost whole but coated in lymphoid tissue and one was in an haemorrhagic area. In the afferent popliteal lymphatic there were about 15 dead, degenerating worms in lymphoid tissue and another eight surrounded by haemorrhage. From the infected right hind leg, a skin lymphatic yielded one dead worm in haemorrhage and a by-pass vessel contained one dead worm in lymphoid tissue. Cat A16 had a microfilarial count of 148/10 mm3 before treatment, which became 0/20 mm3 half an hour after treatment. One hour after treatment the cat died, with blood and saliva pouring from mouth and nose. The lungs were haemorrhagic. DISCUSSION Levamisole showed activity in vitro against the adult worms, infective larvae and microfilariae of B. pahangi, but the dose levels needed to immobilize the worms were very high, compared with the level used in vivo. The infected cats were treated with levamisole at 125 mg/kg. In vitro, however, to immobilize adult worms, infective larvae and microfilariae respectively in 5 mins, 5000, 10000 and 5-10000 mg/kg was the equivalent dosage needed. The LD50 at 24 hours for infective larvae was equivalent to 400 mg/kg and microfilariac were immobilized at 24 hours by the same level of drug. Natarajan et ah (1974) found that all three stages of Breinlia sergenti were susceptible to the drug. The infective larvae and microfilariae became sluggish after 15 mins in a 0-1 % solution, while adult female worms lost motility on contact with a 001 % solution. 25
R. ROGERS and D. A. DENHAM
The increased muscular tone observed in their adultworms with high drug concentrations, was seen with B. pahangi adults, as shown by the hyperactive coiling noted above. Zaman and Natarajan (1973) found that adult Br. sergenti in the slow loris were killed by much higher doses of levamisole than those needed to kill microfilariae (4 x 50 mg/kg killed adults and microfilariae, while doses, on alternate days, of 1-32 mg/kg killed all microfilariae). The same amounts of diethylcarbamazine (DEC) were only partially active against either stage of the worm. Adult B. pahangi in cats are killed by DEC at 50 mg/kg for five days. The LD50 for B. pahangi infective larvae at 24 hours in vitro is 0-2% DEC, compared with 0-04% levamisole (unpublished data). More than 0-2% DEC is required to kill all B. pahangi microfilariae in 24 hours in vitro, whilst 0-04 % levamisole is adequate. 0-3 % DEC and 0-07 % levamisole kill microfilariae in 6 hours. Levamisole is thus effective against all three stages of the parasite at much lower levels than DEC in vitro but the levels are much higher than those which can be used in vivo. The reversible paralysis which Coles and Jenkins (unpublished) noted when adult N. brasiliensis were exposed to 0-01 % levamisole in vitro did not occur with B. pahangi. They postulated that a neuro-receptor in the worm was continually stimulated by the high dose of drug, causing paralysis, but that later the receptor could no longer be stimulated and the worms became motile again. Levamisole was a fairly effective filaricide against the developmental stages of B. pahangi in mosquitoes, even at quite low levels (0-008%). Gerberg et.al. (1972) found that levamisole had a detrimental effect on the development of Dirofilaria immitis in A. aegypti. Although levamisole has some filaricidal activity in vitro and in mosquitoes, it had a limited effect on B. pahangi in cats. Levamisole (but not tetramisole) had an immediate microfilaricidal effect, but some adult female must still have produced microfilariae long after treatment, as the microfilarial level of M42 rose again after initially being reduced to zero. The adult worms from M44 were all alive and undamaged. Some worms from M42 were dead and some females contained abnormal embryos, indicating that perhaps the drug had a much longer-term effect on the worms than could be detected in the cat killed 20 days after treatment (although some worms may have been dead before drug treatment). Mak et ah (1974) found that levamisole hydrochloride in total doses of 30-150 mg/kg body weight had micro- and macrofilaricidal effects on subperiodic B. malayi infections in cats. Zaman and Lai (1973) treated two patients infected with Wuchereria bancrofti with levamisole. A single dose of 120 mg caused a marked reduction in the numbers of circulating microfilariae, but several doses were needed to reduce the count to zero. One patient, after one treatment, developed side effects similar to those occurring after DEC treatment. They concluded that this reaction was due to destruction of the parasites and that caution would be needed in treating patients with high microfilaraemia. Zaman and Fung (1973) successfully treated a case of eosinophilic lung with levamisole. O'Holohan and Zaman (1974) found levamisole to be microfilaricidal in humans infected with B. malayi, but adult worms were not killed or sterilized by the dosages used. Tulloch and Anderson (1972) found levamisole to be prophylactic against D. immitis infections in dogs. Duke (1974) found that levamisole made chimpanzees infected with Onchocerca volvulus ill, at doses usually used for intestinal nematodes. However, when injected intra26
Studies with Brugia pahangi 12
muscularly at 10 mg/kg for 15 days (a total dose of 150 mg/kg or 2-4 g), microfilarial counts became very low and remained at zero after complete clearance of microfilariae with DEC. Thus microfilariae were affected by levamisole and the adult worms were probably killed or sterilized. (The animals were not necropsied, so that the state of the adult worms could not be determined.) Duke (1975) treated O. volvulus in a chimpanzee and a human, giving the drug intramuscularly at 2 and 2-5 mg/kg for up to 15 days. The maximum dose tolerated was 2 mg/kg. No great effect was observed on either microfilariae or adult worms. The main conclusions which can be drawn from the present work are thus: (1) that the in vitro action of levamisole on B. pahangi occurred with higher concentrations than against Br. sergenti and than can be tolerated in vivo. (2) that levamisole is active against the developmental stages of B. pahangi in the mosquito vector. (3) that levamisole is very active against microfilariae in vivo, but less active against adult worms. It appears to be much more active on Br. sergenti in vivo than against B. pahangi and O. volvulus. In view of the above observations and the relatively high toxicity of the drug (shown by the side effects after treatment of cats and the death of one), it would seem that there will be no great potential for levamisole for use in human filariasis. REFERENCES DENHAM, D. A., PONNUDURAI, T., NELSON, G. S., GUY, FRANCES and ROGERS, ROSEMARY (1971) The effect of Metrifonate on Brugia paliangi infections in domestic cats. Bulletin of the World Health Organization, 45, 423-429. DUKE, B. O. L. (1974) The effect of Levamisole on Onchocerca volvulus. Transactions of the Royal Society of Tropical Medicine and Hygiene, 68, 71. DUKE, B. O. L. (1975) Further trial of Levamisole against Onchocerca volvulus. Transactions of the Royal Society of Tropical Medicine and Hygiene, 69, 287. GERBERG, E. J., KUTZ, F. W. and TULLOCH, G. S. (1972) Preliminary evaluation of 1-Tetramisole as an antifilarial in a mosquito test system. In Canine Heartworm Disease. The current knowledge (Ed. R. E. Bradley). Florida, pp 105-108. GRAS, G. (1974) Les anthelminthiques intestinaux; aspects pharmacologiques. Medecine d'Afriqtte Noire, 21,11-28. MAK, J. W., ZAMAN, V. and SIVANANDAM, S. (1974) Antifilarial activity of Levamisole hydrochloride against subperiodic Brugia malayl infection of domestic cats. American Journal of Tropical Medicine and Hygiene, 23, 369-374. NATARAJAN, P. N., ZAMAN, V. and YEOH, T. S. (1974) In vitro activity of Levamisole on the infective larvae, microfilariae and adult worms of Breinlia sergenti. InternationalJournal for Parasitology, 4,207-210. O'HOLOHAN, D. R. and ZAMAN, V. (1974) Treatment of Brugia malayl infection with Levamisole. Journal of Tropical Medicine and Hygiene, T7t 113-115. THIENPONT, D., VANPARIJS, O F. J., RAEYMAEKERS, A. H. M., VANDEBERK, J., DEMOEN, P. J. A., ALLEWIJN, F. T. N., MARSBOOM, R. P. H., NIEMEGEERS, C. J. E.. SCHELLEKENS, K. H. L. and JANSSEN, P. A. (1966) Tetramisole (R8299)—a new potent broad spectrum anthelmintic. Nature (London), 209,1084-1086. TULLOCH, G. S. and ANDERSON, R. A. (1972) Tetramisole and canine dirofilariasis. In Canine Heartworm Disease. Tfte current knowledge. Ed R. E. Bradley). Florida, pp 101-104. VAN den BOSSCHE, H. (1972) Biochemical effects of Tetramisole. In Comparative Biocliemistry ofParasites (Ed H. Van den Bossche). Academic Press: New York, pp 117-125. VAN den BOSSCHE, H. and JANSSEN, P. A. (1967) Mode of action of Levamisole on Ascaris. Life Sciences, 6, 1781-1792. VAN den BOSSCHE, H. and JANSSEN, P. A. J. (1969) The biochemical mechanism of action of the antincmatodal drug Tetramisole. Biochemical Pharmacology, 18,35-42. 27
R. ROGERS and D. A. DENHAM VAN NEUTEN, J. M. (1972) Pharmacological aspects of Tetramisole. In Comparative Biochemistry of Parasites (Ed H. Van den Bossche). Academic Press: New York, pp 101-115. ZAMAN, V. and FUNG, W. P. (1973) Treatment of eosinophilic lung with Levamisole. Transactions of the Royal Society of Tropical Medicine and Hygiene, 67, 144-145. ZAMAN, V. and LAL, M. (1973) Treatment of Wuchereria bancrofti with Levamisole. Transactions of the Royal Society of Tropical Medicine and Hygiene, 67, 610. ZAMAN, V. and NATARAJAN, P. N. (1973) The antifilarial activity of Levamisole on Breinlia sergenti compared with diethylcarbamazine. Journal of Tropical Medicine and Hygiene, 76, 126-128. Accepted 2. October 1975.
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