Chemotherapeutic studies on Litomosoides carinii infection of Mastomys natalensis*

Bull. Org. mond. Santd 11971, 44, 757-763Bull. Wld Hlth Org. Chemotherapeutic Studies on Litomosoides carinil Infection of Mastomys natalensis* 2. The Activity of Drugs against Microfilariae GEORG LAMMLER,l HEIKE HERZOG 2 & HANS RUDOLF SCHOTZE 2 Comparative investigations of the chemotherapeutic activity of various filaricidal compounds and other anthelmintics were made with Litomosoides carinii infections in Mastomys natalensis, special consideration being given to the microfilaricidal activity of the compounds. Diethylcarbamazine, cyclohexanecarboxylic acid-N-methylpiperazide citrate (HOE 28637a), tetrahydropyranecarboxylic acid-N-methylpiperazide citrate (HOE 29691a), (+ )-tetramisole, (- )-tetramisole (levamisole), and metrifonate proved highly effective against microfilariae whereas gentian violet, methylene violet, and the anthelmintics metyridine, disophenol, pyrantel tartrate, methylpyrantel tartrate and parbendazole showed no, or only inadequate, activity. The usefulness of L. carinii infections in M. natalensis is discussed and is recommended as a model for chemotherapeutic screening tests and for the quantitative evaluation of microfilaricidal activity. Since the discovery of the filaricidal activity of diethylcarbamazine by Hewitt et al. (1947) working with cotton rats (Sigmodon hispidus) infected with Litomosoides carinii, no other compounds of prac- tical value have been discovered in the laboratory, although several thousand compounds must have been screened in laboratories of the pharmaceutical industry and elsewhere. Neither antimonial and arsenical compounds nor various non-metallic sub- stances with filaricidal activity in cotton rat filariasis proved to be useful in human therapy or against filarial infections in domesticated animals on a broad scale. The only compound, other than diethyl- carbamazine, widely used in the therapy of onchocer- ciasis is suramin, which emerged as the result of work in the field. But this drug did not show appre- ciable activity against L. carinii in cotton rats (Hawking, 1963, 1966b). * 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. 2 Scientific Assistant, Institute for Parasitology and Para- sitic Diseases of Animals, Justus Liebig University, Giessen, Federal Republic of Germany. 3Supplied by PAG Presswerk, AG, Essen, Federal Repub- lic of Germany. It appeared to be necessary therefore to evaluate various well-known or newly developed drugs for filaricidal activity against microfilariae or adults of L. carinii in Mastomys natalensis and to compare the results with those obtained in filariasis of cotton rats. In a continuation of our investigations (Limmler et al., 1971b), comparative studies on the antiffilarial activity of various well-known or newly developed drugs are described here with special reference to microfilaricidal effectiveness. MATERIALS AND METHODS The present experiments were carried out in M. natalensis bred in this institute and infected with L. carinii, using homogeneous colonies of the rat mite Ornithonyssus bacoti as intermediate hosts. The animals were fed on a diet developed for Syrian hamsters and kept in small groups in Macrolon (polycarbonate) cages.3 Drinking water was available ad libitum. At the end of the prepatent period, the infected animals were examined quantitatively by the method of Raether & Meyerhofer (1967) for microfilariae in the peripheral blood twice before treatment and at weekly or 2-weekly intervals after treatment. 2688 -757 758 G. LAMMLER, H. HERZOG & H. R. SCHUTZE Table 1. Chemotherapeutic efficacy of diethylcarbamazine citrate, HOE 28637a, HOE 29691 a, HOE 33258 diphosphate, and HOE 33258 trihydrochloride against Litomosoides carinii in Mastomys natalensis Dosage No. of Percentage reduction of microfilarial count the following Average no. of (mg/kg animals number of days after the start of treatment a microfilariae at necropsy Encap- x 5) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~sulation Ix Start| End 3 7 14 21 42 70 100 Females1 Males Total Diethylcarbamazine citrate (oral) 25 10 9 93.2 74.2 13.8 0 13.0 0 1 3 1 6 29 0 50 9 8 87.3 68.5 26.2 0 18.8 0 13 14 27 0 60 6 6 95.6 88.7 67.3 44.3 21.9 55.7 9 5 14 0 100 10 10 94.0 85.7 46.8 2.8 35.2 0 8 15 23 0 125 5 4 99.3 95.7 65.0 72.4 29.4 74.0 9 6 15 0 Control 6 6 0 0 0 0 0 0 13 9 22 0 Control 4 4 9.6 10.7 15.2 5.1 1.2 17.6 1 2 8 20 0 Control 4 4 15.6 13.9 0 0 0 3.7 8 6 14 0 HOE 28637a (oral) 25 10 8 78.8 83.5 80.3 44.9 7.1 39.5 50.4 18 8 24 0 50 9 9 81.8 77.4 53.4 0 0 0 0 14 9 23 0 60 6 6 84.2 89.9 57.2 49.8 44.0 41.0 0 12 10 22 0 100 8 5 92.1 91.6 77.2 51.5 47.4 51.1 54.4 13 15 28 0 125 5 4 97.1 90.0 47.4 56.1 0 34.3 13 12 25 0 150 5 5 95.1 88.4 46.0 60.0 46.2 55.5 10 7 1 7 0 Control 4 4 15.6 13.9 0 0 0 3.7 8 6 14 0 HOE 29691a (oral) 50 10 7 85.3 85.3 42.4 0 0 0 0 11 8 19 0 60 5 4 95.8 86.7 60.8 15.4 81.8 81.0 12 4 16 0 100 10 9 97.5 96.4 82.3 51.5 30.2 34.5 71.5 4 2 6 0 Control 5 5 0 0 0 0 0 37.5 45.8 7 6 13 0 Control 4 4 9.6 10.7 15.2 5.1 1.2 20.6 27.6 12 8 20 0 HOE 33258 diphosphate (subcutaneous) 10 6 6 87.1 99.5 98.1 96.7 93.1 162.6 15 9 24 0 Control 7 7 0 0 0 0 14.21 6.6 31 29 60 0 HOE 33258 trihydrochloride (subcutaneous) c 5 5 5 49.8 98.2 94.4 98.2 83.0 80.0 14 15 29 2 2.5 5 4 55.6 99.0 94.8 97.3 91.7 90.7 27 24 51 1 1.25 5 5 31.5 75.3 63.2 78.1 70.6 45.7 14 9 23 1 Control 4 4 15.6 13.9 0 0 0 3.7 8 6 14 0 a The percentage reduction of microfilariae was calculated by taking the average number of microfilariae before the treatment as 100. b Number of animals with macrofilariae encapsulated in fibrin. c This preparation contains an addition of 0.5 % of lidocaine. CHEMOTHERAPY OF LITOMOSOIDES CARINII INFECTION IN MASTOMYS NATALENSIS. 2 Groups of 4-10 animals were treated orally or sub- cutaneously with different dosages of various drugs about 90 days after infection in single daily doses for 5 consecutive days (see Tables 1-3). The animals were necropsied 42, 70 or 100 days after the begin- ning of treatment. Adult filariae were removed from the pleural or peritoneal cavities, counted, and examined for mobility and viability. The reduc- tion in the numbers of microfilariae in treated M. natalensis was evaluated in comparison with the numbers in infected control animals, taking the average number of microfilariae per group before treatment as 100. The following drugs were tested: (1) Diethylcarbamazine; the pure drug was dis- solved in water and administered orally. (2) Cyclohexanecarboxylic acid-N-methylpipera- zide citrate (HOE 28637a, Hoechst). The compound, which was discovered and described by Loewe et al. (1968a, 1968b), was dissolved in water and administered orally. (3) Tetrahydropyranecarboxylic acid-N-methylpi- perazide citrate (HOE 29691a, Hoechst). The sub- stance was dissolved in water and administered orally (see Loewe et al., 1968b). (4) 2-[2-(4-hydroxyphenyl)-6-benzimidazolyl]-6-(1- methyl-4-piperazyl)benzimidazole diphosphate (HOE 33258, Hoechst). The compound was suspended in water and administered subcutaneously (see Raether & Liimmler, 1971; Lammler et al., 1970). (5) (±)-Tetramisole (Citarin, Bayer). The com- pound was available in the form of 10% solution. Doses are expressed in terms of the free base. The aqueous solution was administered orally. (6) Levamisole ((-)-tetramisole) (Imperial Chemi- cal Industries). The compound was dissolved in water and administered orally. (7) Metrifonate (Neguvon, Bayer). The compound was dissolved in water and administered orally. (8) Gentian violet B (Merck), a triphenylmethane dye (see Hawking, 1963). The substance was dis- solved in water and administered subcutaneously. (9) Methylene violet (Chroma-Gesellschaft), a dye belonging to the safranin series (see Hawking, 1963). The dye was dissolved in water and administered subcutaneously. (10) In addition, various other potential anthel- mintics for veterinary use, such as parbendazole (Smith, Kline & French), pyrantel tartrate (Pfizer), methylpyrantel tartrate (Pfizer), metyridine (Cela), and disophenol (Cyanamid) were evaluated for filari- cidal activity. RESULTS The oral administration of diethylcarbamazine in doses of 5 x 25-5 x100 mg per kg of body weight caused a rapid decrease of more than 90% in the microfilarial count of the peripheral blood during therapy. This fall was followed by an increase in the number of microfilariae in the blood to nearly the original levels within 2 weeks of the maximum reduction (Table 1 and accompanying figure). No influence of the drug on the adult parasites could be found. The evaluation of the cycloaliphatic carboxylic acid-N-methylpiperazide derivatives HOE 28637a and HOE 29691a showed that, when administered in the same dosages, they produced very similar microfilaricidal effects and also showed no activity against the mature parasites (Table 1, Fig. 1). The oral administration of the broad-spectrum anthelmintics (±)-tetramisole and (-)-tetramisole (levamisole) in doses higher than 5 x 10 mg/kg a) 200 - 100 -o 50 E ° 10 a Cs 0 uD 1 M~ 0.5 II 0 10 20 30 40 50 Days after start of treatment 60 70 WHO 0114 Fig. 1. Microfilaricidal effectiveness of diethylcarbama- zine, levamisole, metrifonate, HOE 33258 diphosphate, and HOE 28637a in M. natalensis infected with L. carinii. The reduction in number of microfilariae is based on an average number of 100 before treatment. The dosages were as follows: diethylcarbamzine, 5 x 100 mg/kg orally; levamisole, 5 x 10 mg/kg orally; metrifonate, 5 x 200 mg/kg orally; ............. HOE 33258, 5 x 10 mg/kg subcutaneously; HOE 28637a, 5 x 100 mg/kg orally. 759 G. LAMMLER, H. HERZOG & H. R. SCHUTZE produced a rapid fall in the microfilarial count of the circulating blood, also during the treatment. This fall was followed by a gradual increase in the numbers of microfilariae within 2 weeks of the maximum reduction. Some of the heavily infected animals died during this period. The compounds also showed marked activity against adult parasites since in most of the treated groups some immobile or dead fibrin-encapsulated macrofilariae could be found at necropsy (Table 2 and accompanying figure). In squash preparations of some living adult female worms, degenerated embryos and micro- filariae were recognized. The oral administration of metrifonate in dosages ranging from 5xlOO mg/kg to 5x400 mg/kg led to an extremely rapid decrease of more than 98% in the microfilarial count of the circulating blood during the 5-day treatment. This reaction was Table 2. Chemotherapeutic efficacy of orally administered (+)-tetramisole, levamisole, and metrifonate against Litomosoides carinii in Mastomys natalensis Dosage NNo. of Percentage reduction of microfilarial count the following Average no. of (mg/kg animals number of days after the start of treatment a microfilariae at necropsy Encap- x 5) Start End 3 7 14 21 42 70 100 Females Males Total (±)-Tetramisole 8 6 4 57.0 30.1 0 0 0 0.3 0 13 18 31 4 16 6 6 92.2 79.5 37.2 54.8 30.4 61.8 0 19 12 31 6 25 5 4 95.5 98.2 69.3 68.9 48.4 77.9 93.9 2 1 3 2 Control 5 5 0 0 0 31.0 73.9 65.9 69.9 5 3 8 0 Control 5 5 22.6 27.2 28.2 0 |29.1 73.6 75.4 24 7 31 0 Levamisole 5 5 4 85.9 72.5 80.9 52.6 75.3 89.2 3 2 5 1 10 4 4 92.6 85.5 64.6 40.7 43.1 87.9 5 5 10 1 20 5 1 93.7 93.4 83.2 24.7 0 0 30 16 46 1 25 5 3 97.6 94.6 57.8 64.8 48.5 36.6 7 4 11 3 50 5 2 97.5 97.5 67.4 62.2 34.4 3.4 13 6 19 1 Control 3 3 46.1 2.1 0 6.4 0 0 18 14 32 0 Control 4 4 0 0 0 0 0 24.6 29 30 59 0 Control 4 4 15.6 13.9 0 0 0 3.7 8 6 14 0 Metrifonate 25 5 5 0 21.0 3.3 5.2 8.6 0 5 3 8 0 50 5 4 27.5 63.9 48.3 18.4 5.6 0 8 8 16 1 75 5 4 99.3 95.6 75.6 71.6 82.8 58.7 15 11 26 4 100 5 4 98.2 91.6 74.6 37.6 13.7 10.8 2 2 4 0 200 5 4 98.8 84.0 59.4 31.0 12.0 0 9 9 18 0 400 5 4 100.0 97.2 55.0 33.1 0 0 16 9 25 1 Control 5 5 0 0 0 0 0 0 51 27 78 0 Control 3 0 0 0 17.3 30.2 32.2 2 3 5 0 Control 4 4 15.6 | 13.9 0 0 0 3.7 8 6 14 0 a The percentage reduction of microfilariae was calculated by taking the average number of microfilariae before the treatment as 100. b Number of animals with macrofilariae encapsulated in fibrin. 760 CHEMOTHERAPY OF LITOMOSOIDES CARINII INFECTION IN MASTOMYS NATALENSIS. 2 Table 3. Chemotherapeutic efficacy of gentian violet, methylene violet, parbendazole, pyrantel tartrate, and methylpyrantel tartrate against Litomosoides carinhl in Mastomys natalensis Dosage No. of Percentage reduction of microfilarial count the following Average no. of (mg/kg animals numbers of days after the start of treatment"a microfilariae at necropsy Encap-b x 5) Start End 3 7 14 21 42 70 100 Females saMales oTotal n Gentian violet (subcutaneous) 0.5 5 5 0 0 0 0 0 16 9 25 0 1.0 5 5 0 0 0 0 0 14 11 25 0 2.0 5 5 0 0 0 0 0 16 9 25 0 Control 3 3 0 40.7 0 0.9 54.3 14 7 21 0 Methylene violet (subcutaneous) 0.5 6 5 5.7 15.6 2.1 75.5 60.0 83.9 87.5 2 0.6 2.6 3 1.0 6 5 35.3 14.9 28.4 67.7 61.2 70.7 85.0 6 5 11 2 2.0 6 6 0 0 9.1 66.6 19.0 48.6 77.0 9 4 13 4 4.0 5 5 24.1 18.3 29.6 25.6 4.2 76.6 80.2 9 6 15 2 Control 5 5 22.6 27.2 28.2 0 29.1 79.6 75.4 24 7 31 0 Control 2 2 0 15.5 19.6 47.3 0 60.3 55.4 3 3 6 1 Parbendazole (oral) 200 5 5 33.8 37.4 43.3 62.0 89.1 97.0 12 1 13 3 400 5 5 7.8 54.4 28.3 51.6 84.2 55.2 11 9 20 5 Pyrantel tartrate (oral) 40 5 1 5 44.1 10.11 8.3 0 0 0 0 56 27 83 3 Methylpyrantel tartrate (oral) 40 5 4 16.9 21.5 4.4 22.7 0 0 0 40 39 79 2 Control 5 4 0 0 0 0 0 0 0 51 27 78 0 a The percentage reduction of microfilariae was calculated taking the average number of microfilariae before the treatment as 100. b Number of animals with macrofilariae encapsulated in fibrin. followed by a steep increase in numbers within the next week and by a gradual approach to the micro- filarial count of the non-treated control group. No activity against adult parasites was noticed at necropsy, except that a few encapsulated worms in the 5 x 400 mg/kg dosage group were seen. The treatment with gentian violet of M. natalensis infected with L. carinii proved to be completely ineffective. The administration of methylene violet was followed by a gradual reduction of the micro- filariaemia but a similar reaction was noticed also in the non-treated control group. The activity of the compound against mature parasites was remark- able because numerous immobile or dead macro- filariae encapsulated in fibrin were found at necropsy. It can be concluded, however, that methylene violet possesses insufficient activity against microfilariae and mature parasites for therapeutic use. The various other anthelmintics such as metyridine, disophenol, pyrantel tartrate, methylpyrantel tar- trate, and parbendazole showed only low or moder- ate activity against microfilariae or adult parasites and are not considered to be of practical conse- quence. 761 G. LAMMLER, H, HERZOG & H. R. SCHUJTZE DISCUSSION The use of a standardized laboratory infection for chemotherapeutic trials of new compounds is an important procedure in the field of experimental filariasis. The introduction of cotton rat filariasis as a model infection more than 25 years ago marked a great progress. Since the discovery of diethylcar- bamazine and related compounds, only few classes of chemicals, with the exception of antimonials and arsenicals, have proved to be effective in laboratory trials. Some of these compounds, such as methylene violet, phenanthridine, and antimitotic compounds, revealed promising activity against L. carinii in cot- ton rats but they did not show sufficient activity against other species of filaria in dogs or man. On the other hand, suramin proved very effective against Onchocerca in man without showing any activity against cotton rat filariasis (Hawking, 1963, 1966a, 1966b). It appeared useful therefore to compare the filari- cidal activities of various well-known or newly devel- oped drugs in L. carinii infections of M. natalensis. The results demonstrate the high, and more-or-less equal, microfilaricidal efficacy of diethylcarbamazine and the similar compounds, cyclohexanecarboxylic acid-N-methylpiperazide citrate and tetrahydropy- ranecarboxylic acid-N-methylpiperazide citrate (HOE 28637a, HOE 29691a), discovered and described by Hewitt et al. (1947) and Loewe et al. (1968a, 1968b), respectively. The potential filaricidal activity of (±)-tetrami- sole, levamisole, and metrifonate might be of great interest in spite of the fact that relatively high doses were necessary to produce an effect. It therefore seems of value to investigate the filaricidal effective- ness of these drugs in comparison with diethylcarba- mazine against other species of filaria in animals and man. The broad-spectrum anthelmintic levamisole, especially, should be analysed carefully because it is also strongly active against Angiostrongylus infec- tions in dogs, unlike the compound HOE 28637a (Liimmler et al., 1971a). To compare the results of these studies with those obtained from the treatment of the L. carinii infec- tions with the basically substituted 2,6-bis-benzimi- dazole derivative HOE 33258 (Lammler et al., 1970b), the microfilaricidal effect of 5 x 10 mg/kg subcutaneous dosages of HOE 33258 is included in Table 1 and the accompanying figure. The results demonstrate the superior microfilaricidal activity of HOE 33258 as regards the intensity of activity and the duration of effect. Similarly, considerable differences between diethylcarbamazine and HOE 33258 diphosphate were found also in L. carinii infec- tions of cotton rats (Raether & Lammler, 1971). In further studies, the remarkable effect of HOE 33258 diphosphate against Dirofilaria immitis and other species of filaria in monkeys should be taken into consideration (Lammler et al., 1971b). The present comparative studies on the chemo- therapeutic activity of drugs against microfilariae demonstrate the usefulness of the L. carinii infection of M. natalensis for chemotherapeutic screening tests, as well as for a quantitative evaluation of the microfilaricidal efficacy of drugs. oUME ETUDES SUR LA CHIMIOTHERAPIE DE L'INFECTION PAR LITOMOSOIDES CARINII CHEZ MASTOMYS NATALENSIS: 2. ACTIVITE DE MEDICAMENTS CONTRE LES MICROFILAIRES Divers composes filaricides et des anthelminthiques ont fait l'objet d'essais comparatifs destines a e'valuer leur activite chimiotherapeutique contre les microfilaires. On a choisi comme modele experimental l'infection de Mastomys natalensis par Litomosoides carinii. La diethylcarbamazine, le citrate de (cyclohexyl- carbonyl)-1 m6thyl4 piperazine (HOE 28637 a), le citrate de (tetrahydropyranecarbonyl)-2 methyl4 piperazine (HOE 29691 a), le (±)-t6tramizole, le (-)-tetramizole et le metrifonate se revelent remarquablement actifs contre les microfflaires. Par contre, le violet de gentiane, le violet de methylene, la metyridine, le disophenol, le tar- trate de pyrantel, le tartrate de methylpyrantel et le parbendazole ne font preuve que d'une efficacite nulle ou tres insuffisante. 762 CHEMOTHERAPY OF LITOMOSOIDES CARINII INFECTION IN MASTOMYS NATALENSIS. 2 763 REFERENCES Hawking, F. (1963) Chemotherapy of filariasis. In: Schnitzer, R. J. & Hawking, F., ed., Experimental chemotherapy, New York, Academic Press, vol. 1, pp. 893-912 Hawking, F. (1966a) Chemotherapy of filariasis. In: Schnitzer, R. J. & Hawking, F., ed., Chemotherapy, New York, Academic Press, vol. 4, pp. 487-493 Hawking, F. (1966b) Chemotherapy in filariasis In: Jucker, E, ed, Progress in drug research, Basel, Birk- hauser, vol. 9, 191-222 Hewitt, R., Kushner, S., Stewart, H., White, E., Wallace, W. S. & Subba-Row, Y. (1947) J. Lab. clin. Med., 32, 1314 Limmler, G. et al. (1971a) Berl. Munch. tierdrzul. Wschr. (in press) Lammler, G. et al. (1971b) Bull. Wld Hith Org., 44, 751 Loewe, H. et al. (1968a) British Patent No. 1, 127, 457 Loewe, H. et al. (1968b) In: Proceedings of the Second International Symposium on Pharmaceutical Chemistry, Miinster Raether, W. & Lammler, G. (1971) Ann. trop. Med. Parasit. (in press) Raether, W. & Meyerhofer, W. (1967) Z. Tropenmed. Parasit., 18, 99-108 4