Chemotherapeutic studies on Litomosoides carinii infection of Mastomys natalensis*

Bull. Org. mond. Sante 1971, 44, 751-756 Bull. Wld Hlth Org. Chemotherapeutic Studies on Litomosoides carinli Infection of Mastomys natalensis * 1. The Filaricidal Action of 2,6-bis-Benzimidazoles GEORG LAMMLER,' HEIKE HERZOG,2 EBERHARD SAUPE2 & HANS RUDOLF SCHOTZE 2 The antifilarial action of 2-[2- (4-hydroxyphenyl)-6-benzimidazolyl]-- (J-methlyl-4- piperazyl)benzimidazole (HOE 33258) was investigated in Mastomys natalensis infected with Litomosoides carinii. The subcutaneous administration of HOE 33258 in a single daily dose for S consecutive days, or at other intervals, produced, depending on the dosage, a rapid reduction in the number of microfilariae in the circulating blood. The reduction amounted to more than 90 % within 7-14 days after the treatment was started or at the end of the dosage schedule. The small, slow increase in the microfilarial count during a period of 6-7 weeks after treatment ended reached not more than half the number present before treatment. HOE 33258 showed marked activity on the reproductive system of mature female worms, although only few macrofilariae were killed by the drug. The results also demonstrated the usefulness of L. carinii infection of M. natalensis as a model for the evaluation of the filaricidal activity of drugs. In the search for new filaricidal compounds, sys- tematic screening tests have been carried out during the past 25 years, almost entirely on Litomosoides carinii in cotton rats (Sigmodon hispidus). Several investigations have provided useful information in relation to the biology of untreated infections as well as to the effects of different types of chemical compounds in naturally acquired or experimental filariasis in cotton rats. In extensive studies on the susceptibility of Masto- mys natalensis to parasitic helminths (Texdorf, 1967; Zahner, 1967; Liimmler et al., 1968b, 1969, 1970), this animal proved to be an excellent final host also for the cotton rat filaria, L. carinii (Limmler et al., 1968a, 1969; Pringle & King, 1968; Zahner et al., 1970a, 1970b). It was therefore of great interest to evaluate the activities of various well-known or newly developed filaricidal drugs against microfilariae or adult parasites in this new laboratory animal. In a recent publication, Foster et al. (1969) reported, for * This project was carried out with the aid of a grant from the World Health Organization. 1 Professor of Parasitology and Director of the Institute for Parasitology and Parasitic Diseases of Animals, Justus Liebig University, Giessen, Federal Republic of Germany. ' Scientific Assistant, Institute for Parasitology and Para- sitic Diseases of Animals, Justus Liebig University, Giessen, Federal Republic of Germany. the first time, the usefulness of the L. carinii infection of M. natalensis for chemotherapeutic experiments. This paper describes an investigation on the anti- filarial activity of 2-[2-(4-hydroxyphenyl)-6-benzimi- dazolyl]-6-(1-methyl-4-piperazyl)benzimidazole(HOE 33258),3 which was discovered and described by Loewe et al. (1967), Lammler & Raether (1968) and Loewe & Urbanietz (1968), working with cotton rats infected with L. carinii. The compound, which appears to be also a useful fluorochrome for staining animal-cell nuclei (Herzog & Schutze, 1968; Lammler & Schutze, 1969), has been found to be one of the most effective filaricidal substances of a series of basically substituted bis- benzimidazoles (Loewe et al., 1967; Loewe & Urbanietz, 1968; Raether & Lammler, 1971). MATERIALS AND METHODS For propagating the L. cariniiinfection in M. natal- ensis, homogeneous colonies of the rat mite Orni- thonyssus bacoti were infected with the larval stages of L. carinii by allowing them to feed on several 3Preparation HOE 33258 was supplied by Farbwerke Hoechst AG, Frankfurt am Main, Federal Republic of Germany. 2687 751- G. LAMMLER AND OTHERS cotton rats or M. natalensis which had numerous microfilariae in their peripheral blood. The animals used in this study were bred by conventional methods in a small breeding station at the Institute; they were fed on a diet developed for Syrian hamsters and kept in small groups in Macrolon cages.' Drink- ing water was available ad libitum. At the end of the pre-patent period, the infected animals were examined quantitatively for microfilariae in the peri- pheral blood twice before treatment and thereafter at weekly or 2-weekly intervals for 70 or 100 days, using the method of Raether & Meyerhofer (1967). 1 Polycarbonate cages, supplied by PAG Presswerk AG, Essen, Federal Republic of Germany. Groups of 6-10 animals were treated with HOE 33258 in form ofthe diphosphate suspended in water, the trihydrochloride dissolved in water, or the free base suspended in oil, in single subcutaneous doses daily for 5 consecutive days, starting about 90 days after infection. The animals were necropsied 70 or 100 days after the beginning of treatment. The adult filariae were removed from the pleural and peritoneal cavities, counted, and examined for mobility and viability. The reduction in numbers of microfflariae in treated M. natalensis was evaluated in compari- sons with infected control animals, the average number of microfilariae per group before treatment being taken as 100. Table 1. Chemotherapeutic activity of subcutaneous administration of HOE 33258 as the diphosphate, the trihydrochloride, and as the free base against Litomosoides carinii in Mastomys natalensis Dosage No. of Percentage reduction of microfilarial count the following Average no. of macrofilariae(mg/kg animals number of days after the start of treatment :a at necropsy Encap- x 5) Start End 3 7 14 21 42 70 100 Females Males Total s Diphosphate salt suspended in water 2.5 6 6 92.8 89.0 61.0 62.2 60.3 47.5 13 1 1 24 0 5 6 6 98.1 97.1 82.5 92.3 93.3 88.1 12 8 20 0 10 6 6 87.1 99.5 98.1 96.7 93.1 62.6 1 5 9 24 0 20 6 6 99.8 100 99.9 99.5 99.3 95.1 26 43 69 0 40 1 0 8 72.4 98.2 99.8 99.8 98.7 93.9 79.9 24 17 41 5 60 1 1 9 73.9 98.5 99.8 99.6 92.9 79.5 53.4 25 1 6 41 7 Control 7 7 0 0 0 0 14.2 6.6 31 29 60 0 Co ntrol 4 3 9.6 23.4 15.2 5.2 1.2 27.6 12 8 20 0 Trihydrochloride salt dissolved in water 10 6 6 64.0 80.0 88.0 84.0 67.0 72.0 2 2 4 0 20 4 3 96.2 99.3 97.6 84.5 85.9 72.6 25 37 62 0 40 1 0 9 80.4 93.0 100 100 67.0 42.6 17.3 1 7 1 4 31 6 60 10 8 84.7 97.3 100 98.7 99.0 93.7 82.0 6 5 1 1 8 Control 6 6 0 0 0 0 0 0 0 13 9 22 0 Free base suspended in oil 2.5 10 9 0 0 9.3 0 2.6 0 0 1 0 10 20 0 5 1 0 1 0 0 43.1 0 0 0 0 0 1 6 1 3 29 0 10 1 0 1 0 13.1 89.2 91.3 50.3 42.9 32.6 30.3 7 6 1 3 0 20 1 0 8 0.1 0 87.0 68.3 67.5 24.6 29.0 1 6 1 0 26 0 40 1 0 1 0 47.1 99.0 96.9 97.1 93.2 90.5 84.0 1 6 34 50 8 Control 6 6 0 0 0 0 0 0 0 13 9 22 0 a The percentage reduction of microfilariae was calculated on the basis of the average number of microfilariae before the treatment. b Number of animals with macrofilariae encapsulated in fibrin. 752 CHEMOTHERAPY OF LITOMOSOIDES CARINII INFECTION IN MASTOMYS NATALENSIS. 1 RESULTS Efficacy of HOE 33258 against microfilariae The subcutaneous administration of HOE 33258, using the aqueous suspension of the diphosphate in single daily doses as low as 2.5 mg per kg of body weight during 5 consecutive days, produced a marked reduction in the number of microfilariae in the peri- pheral blood. A maximum decrease was noticed between the third and the seventh days from the beginning of treatment (Table 1, Fig. 1). This reaction was followed by an increase of the micro- filarial count to half the number present before treatment. The higher dosages between 5 x 10 mg/kg and 5 x 60 mg/kg given subcutaneously produced falls in the microfilarial count of more than 90% for 42 days or longer, but even these dosages did not remove the microfilariae from the circulating blood completely and the same dosages did not prevent a subsequent increase in number. The maxi- mum reduction in numbers of microfilariae was observed about 14 days from the beginning of treat- ment with 5 x 40 mg/kg and 5 x 60 mg/kg, given subcutaneously. The treatment of the L. cariniiinfection in M. natal- ensis with equal doses of the compound in the form of the soluble trihydrochloride produced a similar effect, whereas the injection of the same dosages in 0 ._ 0 0 .E 0 C; D: 200 100 50 10 5 0.5 0.1 0 10 20 30 40 50 Days after start of treatment 60 70 WHO 01149 Fig. 1. Microfilaricidal effectiveness of HOE 33258 diphosphate in M. natalensis infected with L. carinii after the administration of 5 consecutive daily sub- cutaneous doses. The reduction in number of micro- filariae is based on an average number of 100 before treatment. The dosages were as follows: C: 5x2.5 mg/kg; D: 5x5 mg/kg; E: 5x10 mg/kg; F: 5x40 mg/kg; G: 5x60 mg/kg. Curves A and B represent the control groups for groups C-D-E and F-G, respectively. the form of the free base suspended in oil showed an irregular and inadequate activity. The micro- filaricidal effect of the water-soluble or water- suspended salts of the compound was more or less proportional to the dose applied. The subcutaneous administration for 5 days of the substance in the form of the diphosphate in the same daily dose of 10 mg/kg, but at different inter- vals (Fig. 2), caused also a considerable fall in the microfilarial count. A maximum reduction of more than 95% in the number of microfilariae in the circulating blood could be observed, depending on the schedule, a few days after the treatment had ceased. Even these regimens did not prevent a slight increase again in the numbers of microfilariae after various intervals. Efficacy of HOE 33258 against macrofilariae The administration of HOE 33258 in the form of the two salts or as the free base suspended in oil did not reveal any marked macrofilaricidal activity. Only when high dosages were applied subcutane- ously (5 x 40 mg/kg and 5 x 60 mg/kg) was a small number of adult parasites killed and encapsulated in fibrin (Table 1). No difference in the activity of HOE 33258 could be seen between males and females of L. carinii. Investigations on the viability of macrofilariae and on the production of microfilariae by the females 0 *8 200 0 =- ioo 20 .2 so E 0 ° 10 5 ._ .2-0 I 0 10 20 30 40 50 Days after start of treatment 60 70 whO 01150 Fig. 2. Microfilaricidal effectiveness of HOE 33258 diphosphate in M. natalensis infected with L. carinii after the administration of 5 daily subcutaneous doses at different intervals. The reduction in number of microfilariae is based on an average number of 100 before treatment. The dosage was 5 x 10 mg/kg. _ -C= 2 = = i B~~~~ ;1Y ~~~~~~~~~ Doys of treatment 1 7 14 21 28C Control group !ltttAB4 8 1 c 8 1216 753 G. LIAMLER AND OTHERS Table 2. Viability of macrofilariae and production of microfilariae after treatment a with a preparation of HOE 33258 b Results at necropsy the following no. of days after the start of treatment: 7 14 21 28 35 49 63 Macrofilariae c +++ +++ +++ ++ +++ +++ + Microfilariae c ++ + ++ +++ +++ +++ ++ Embryos c +++ + ++ +++ +++ +++ ++ Average no. of living microfilariae in culture 764 826 580 2 020 1 906 2 100 3 220 Treated groupsC Percentage reduction of microfilariae in blood: individual groups 95.6 96.4 100 99.8 87.1 94.0 75.0 no. of animals 21 18 15 12 9 6 3 total 99.5 99.1 99.9 99.1 90.1 83.0 59.6 Macrofilariae +++ +++ +++ +++ +++ +++ +++ Microfilariae +++ +++ +++ +++ +++ +++ +++ Embryos c +++ +++ +++ +++ +++ +++ +++ Average no. of living microfilariae in culture 8 086 8 053 2 080 13 755 7 750 9 560 6 210 Control groups e Percentage reduction of microfilariae in blood: individual groups 0 0 0 0 0 0 0 no. of animals 21 18 15 12 9 6 3 total 0 0 0 0 0 0 0 Percentage reduction in numbers of micro- filariae produced by female worms of treated animals in vitro 90.6 89.8 72.2 85.4 75.5 78.1 48.2 a Dosage: 5 x 20 mg/kg subcutaneously. b This formulation of the trihydrochloride contained 0.5 % of lidocaine. c + = Large proportion of immobile or dead macrofilariae, microfilariae or embryos; ++ = some immobile or dead macrofilariae, micro- filariae or embryos; + + + = normal populations of living macrofilariae, microfilariae and embryos. d A few macrofilariae were found to be encapsulated in fibrin. c A total of 3 animals per group and per week. after treatment with HOE 33258 in subcutaneous doses of 5 x 20 mg/kg demonstrated that the repro- duction rate of female worms was markedly de- creased (Table 2). The production of microfilariae by 2 female worms isolated from the treated animals and incubated in 0.8 ml of phosphate-buffered saline (pH 6.5) and 0.2 ml of Mastomys serum at 370C for 24 hours was considerably reduced for several weeks compared with the number of microfilariae produced by 2 females from untreated animals under the same conditions. This long-term effect appears to be related to the large reduction in numbers of circulating microfilariae in the blood of the host. Microscopical examination of the reproductive sys- tem of female worms separated from treated animals showed numerous deformed and degenerated embryos 2, 3, and 9 weeks after the beginning of the treatment. DISCUSSION The subcutaneous administration of HOE 33258 in the form of the diphosphate suspended in water in a single daily dose of 2.5 mg/kg for 5 consecutive days produced a rapid decrease of more than 90% in the microfilarial count in the circulating blood during the first 7 days after the beginning of treat- ment. Higher doses of the compound administered in the same schedule intensified the reduction in number of microfilariae gradually by more than 99% 754 CHEMOTHERAPY OF LITOMOSOIDES CARINII INFECTION IN MASTOMYS NATALENSIS. 1 between 14 and 21 days. After this period, a reduc- tion in the number of microfilariae of more than 90% was observed to last for 6 weeks or longer (Fig. 1, Table 1). The results demonstrate an impor- tant difference in the effectiveness of the basically substituted 2,6-bis-benzimidazoles in comparison with that of diethylcarbamazine (Limmler et al., 1971). The latter compound, in an oral dosage 10 times as high as the subcutaneous dose of HOE 33258 administered in the current tests, caused a similar, but smaller, reduction in the number of microfilariae in the circulating blood and this fall was followed by a rapid rise within 2 weeks. When HOE 33258 was administered subcutane- ously in 5 daily doses of 10 mg/kg at days 1, 3, 5, 7, 9, days 1, 4, 8, 12, and 16 or days 1, 7, 14, 21, and 28, the fall in the microfilarial count was slower, depending on the schedule, and the maximum reduc- tion of more than 95% could always be observed directly after the treatment ended (Fig. 2). The fact that the subsequent increase in number of microfilariae in the circulating blood after treat- ment never reached half the number before the treatment might be due either to the amount of HOE 33258 in the blood and tissues or to the action of the drug on the reproductive system of female worms. The strong depression of microfilarial production by female worms isolated from treated animals in weekly or 2-weekly intervals and kept in in vitro culture for 1 day (Table 2) demonstrated the influ- ence of the drug also upon the mature parasites. The similarity between the high percentage reduc- tion of microfilariae for 7-9 weeks in the circulating blood and the lowering of the microfilarial produc- tion by treated female worms in in vitro culture after the administration of HOE 33258 trihydrochloride in daily subcutaneous doses of 20 mg/kg for 5 con- secutive days (Table 2) proved that HOE 33258 affected mature worms, although only a few dead macrofilariae encapsulated in fibrin could be found. The irregular and inadequate activity of the com- pound in the form of the free base suspended in oil might depend on slow or irregular absorption or on local irritation of the tissues. The results of these studies of L. carinii infection in M. natalensis have demonstrated that, in com- parison with infections in cotton rats, there are some differences (Raether & Liimmler, 1971). The subcutaneous administration of HOE 33258 diphos- phate in the same dosages to cotton rats infected with L. carinii not only produced a higher percen- tage reduction of microfilariae in the circulating blood but also showed a stronger activity against adult parasites. Such an effect could be observed after the subcutaneous administration of doses as low as 5 x 4 mg/kg and 5 x 8 mg/kg. The higher susceptibility of macrofilariae in cotton rats, espe- cially of the female worms, was linked with a strong action of the drug on the reproductive system, as well as with the deaths ofworms and their subsequent encapsulation in fibrin (Raether & Limmler, 1971). HOE 33258 was found to persist for a long time in the tissues of cotton rats and M. natalensis treated with the drug. Fluorescence microscopical studies of blood-smears and tissue sections from treated animals revealed bright fluorescence of the cell nuclei of microfilariae as well as of the blood leucocytes and cell nuclei in host tissue and organs. In smears and histological sections of liver and kidney, relatively strong fluorescence of the nuclei could be recognized up to 37 days after treatment, or longer (Liimmler & Schutze, 1969). However, it cannot be concluded from this observation that the filaricidal effect over a period of several weeks depends entirely on the presence of HOE 33258 in cell nuclei of host tissues and organs. It seems that both the action of the drug on female worms and the residual HOE 33258 in the tissues and organs together influenced the numbers of microfilariae in the circulating blood. The present studies on the filaricidal activity of HOE 33258 in M. natalensis infected with L. carinii confirmed the excellent results obtained in chemo- therapeutic experiments made previously on cotton rat filariasis (Lammler & Raether, 1968; Raether & Limmler, 1971). The compound proved to be highly effective against microfilariae and showed a marked influence on the reproduction rate of mature female worms. Furthermore, the results demon- strated that L. carinii infection in M. natalensis is a useful and valuable model for evaluating the chemo- therapeutic activity of filaricidal drugs in laboratory experiments. The effectiveness of HOE 33258 against other filaria species was also recognized. The subcutane- ous administration of the compound in the form of the trihydrochloride dissolved in water was highly effective against Dirofilaria immitis in a dog, against Tetrapetalonema marmosetae (Faust, 1935) in two tamarins (Saguinus oedipus), and against an uniden- tified species of microfilaria in a gorilla (Liammler, Schuitze & Herzog, unpublished data), without any side-effects. 755 756 G. LAMMLER AND OTHERS REFERENCES Foster, R. et al. (1969) Ann. trop. Med. Parasit., 63, 95-107 Herzog, A. & Schuitze, H. R. (1968) Dtsch. tierdrztl. Wschr., 75, 476 Lammler, G. & Raether, W. (1968) Chemotherapeutic experiments with a 2,6-bis-benzimidazole in cotton rats infected with Litomosoides carinii. In: Proceedings of the 8th International Congresses of Tropical Medicine and Malaria, Teheran, p. 139 Lammler, G. & Schutze, H. R. (1969) Naturwissen- schaften, 56, 286 Lammler, G. et al. (1968a) Z. Parasitenk., 30, 281-290 Lammiler, G. et al. (1968b) Z. Parasitenk., 31, 166-202 Lammler, G. et al. (1969) Z. Parasitenk., 32, 254-265 Lammler, G. et al. (1970) Br. vet. J., 126, 427-439 Loewe, H. & Urbanietz, J. (1968) 2,6-bis-benzimidazoles, a new class of anthelmintics. In: Proceedings of the 8th International Congresses on Tropical Medicine and Malaria, Teheran, p. 139 Loewe, H. et al. (1967) Netherlands Patent No. 6704539 Pringle, G. & King, D. F. (1968) Trans. roy. Soc. trop. Med. Hyg., 62, 11 Raether, W. & Ldmmler, G. (1971) Ann. trop. Med. Parasit. (in press) Raether, W. & Meyerhofer, W. (1967) Z. Tropenmed. Parasit., 18, 99-108 Texdorf, J. (1967) Mastomys natalensis (Smith, 1834) und ihre Eignung als Endwirt fur Trematoden im Ver- gleich zu anderen Laboratoriumstieren. Inaugural Dis- sertation, Justus Liebig University, Giessen Zahner, H. (1967) Infektionsversuche mit Darmnematoden und Cestoden bei Mastomys natalensis (Smith, 1834) im Vergleich zu anderen Laboratoriumstieren. Inaugu- ral Dissertation, Justus Liebig University, Giessen Zahner, H. et al. (1970a) Z. Parasitenk., 33, 267-281 Zahner, H. et al. (1970b) Z. Parasitenk., 33, 128-140 RE-SUME ETUDES SUR LA CHIMIOTHERAPIE DE L'INFECTION PAR LITOMOSOIDES CARINII CHEZ MASTOMYS NATALENSIS: 1. ACTION FILARICIDE D'UN BIS-BENZIMIDAZOLE-2,6 On a cherche 'a determiner l'activite antifilarienne du diphosphate de [(hydroxy-4 phenyl)-2 benzimida- zolyl-6]-2 (methyl-1 pipdrazyl-4)-6 benzimidazole (HOE 33258) chez Mastomys natalensis infecte par Litomosoides carinii. Administre quotidiennement par voie sous-cutande a la dose de 2,5 mg par kilo de poids corporel pendant 5 jours consecutifs, ou 'a des doses superieures et a inter- valles plus longs, le compose entraine une diminution rapide du nombre des microfilaires dans le sang circulant. La reduction de la densite microfilarienne depasse 90% dans les 7 a 14 jours suivant le debut du traitement. Les numerations pratiquees 6 a 7 semaines apres le traitement ne montrent qu'une reapparition lente et moderee des microfilaires dont le nombre reste inferieur de moitie au nombre initial. Bien que ne provoquant qu'une faible mortalite parmi les macrofilaires, le HOE 33258 agit de fagon nette sur la fonction de reproduction chez le ver femelle adulte. Cette etude Imontre que l'infection de M. natalensis par L. carinii peut servir de modele experimental pour l'evaluation de l'activite filaricide de composes chimiques.