The effect of diethylcarbamazine in a murine model of Brugia malayi microfilaraemia*

BRIEF COMMUNICATIONS Bulletin of the World Health Organization 57 (2): 329-330 (1979) The effect of diethylcarbamazine in a murine model of Brugia malayi microfilaraemia * M. NEILL' & J. W. KAZURA 2 Abstract The effect of diethylcarbamazine was tested in a murine model of Brugia malayi microfilaraemia. A course of therapy similar to that used to treat human infection led to more than a 90% decrease in circulating parasites. Experiments in which different amounts of diethylcarbamazine were given as a single dose indicated that its microfilaricidal activity is dose- dependent. The animal model of B. malayi micro- filaraemia may be usefulfor studies of the mechanism of action and pharmacology of diethylcarbamazine and may be applied to the screening of new micro- filaricidal drugs. Most studies of filaricidal drug efficacy have been based on the effect of these agents on parasites not pathogenic for man, such as Litomosoides carinii in the cotton rat (1). Although this model has been useful, testing these drugs against parasites pathogenic to man might provide information more relevant to their activity in human disease. The development of persistent microfilaraemia in laboratory mice injected intravenously with Brugia malayi microfilariae provides a model that may be useful for the assessment of microfilaricidal drugs. Animals injected intravenously with B. malayi microfilariae exhibit a peak microfilaraemia within 7-10 days; the counts level off for approximately 50 days and then progressively decline. In this paper we describe the effect of diethylcar- bamazine, a drug commonly used in the treatment of several forms of human and animal filariasis,' in mice injected with B. malayi microfilariae. * From the Department of Medicine, Case Western Reserve University and University Hospitals, Cleveland, OH 44106, USA. This study was supported by the US Public Health Service, grant AI-15351 and the Rockefeller Foundation, grant GAHS-7704. 'Resident. 2Assistant Professor of Medicine, Division of Geographic Medicine. aReport of the first meeting of the Scientific Working Group on Filariasis. WHO unpublished document TDR/MPD/SWG-FIL (1)/ 77.3. Materials and methods Microfilariae were obtained from chronically in- fected Mongolian jirds.b The animals were lightly anaesthetized with thiopental sodium and 20 ml of Dulbecco's phosphate buffer was instilled in- traperitoneally via an 18-gauge plastic catheter. After massaging the abdomen, the fluid containing microfilariae was withdrawn. The microfilariae were washed three times (100 g for 10 min at 22°C) and resuspended to a final concentration of 2 x 101 organisms per millilitre of phosphate buffered saline, pH 7. Female CFI mice weighing 18-20 g were each injected intravenously with 0.5 ml of this suspension, and at various intervals thereafter 80 Iul of blood was drawn from the retro-orbital plexus of each of the injected mice (between 11 h 00 and 13 h 00). The red cells were lysed with distilled water and the microfilariae were then counted in Sedgewick-Raf- ter chambers.c Diethylcarbamazine was supplied as the citrate salt. The drug was dissolved in sterile distilled water immediately before use and each dose was adminis- tered in a volume of 0.2 ml by oesophageal intuba- tion. The level of circulating microfilariae was fol- lowed every 2-3 days during therapy and for 3 weeks thereafter. Results Two groups each of 10 mice, injected 4 weeks earlier with microfilarial suspension, had mean mi- crofilarial counts of 55±7 per 100 RI blood. One group of mice received 6 mg of diethylcarbamazine per kg of body weight daily while the other group received sterile water. Animals given the drug had only 34% of the microfilarial level of the controls after 24 h (P< 0.05). The levels remained low (3-19% of control levels) for the duration of therapy and following its completion (Fig. 1). bObtained from Dr J. McCall, University of Georgia, Athens, GA, USA. c Curtin-Matheson, Cleveland, OH, USA. 3804 329- 330 BRIEF COMMUNICATIONS CE, z :) 100 0 0 < 80 <0 60 00 O 40'40 m 20 U- 0 co v I lH < 0 1 3 5 7 9 11 17 30 w fr .TIME (DAYS)0 DEC 6 mg / kg per day Fig. 1. The effect of a 10-day course of diethylcar- bamazine (DEC) on the level of circulating B. malayi microfilariae. Since the microfilaricidal capacity of diethylcar- bamazine was evident within 24 h, the effect of single-dose therapy and the response to lower doses was evaluated. Four groups each of five mice were given a single dose of 0.187, 0.375, 1.5, and 6 mg/kg body weight; after 24 h the percentage reduction in the microfilarial count was, respectively, 0, 30, 52.6, and 66 (Table 1). The treated mice were then followed every 2-3 days for 3 weeks; the numbers of circulating microfilariae were not significantly diffe- rent from those present 24 h after therapy. Table 1. The effect of diethylcarbamazine administered in a single dose on the level of circulating B. malayi microfilariae in CF1 mice Dose Microfilariae per 100 Ril blood(mg/kg P value body weight) Pretreatment Post-treatment 0.187 50.0 ± 2.2a 50.8 ± 8.6 > 0.9 0.375 50.0 ± 2.2 a 35.0 ± 4.6 < 0.05 1.5 50.0 ± 2.2 a 23.7 ± 8.7 < 0.05 6.0 72.0 ± 7.6 24.4 ± 1.8 <0.05 a A pool of infected mice was used for this experiment. Discussion The murine model of B. malayi microfilaraemia employed in the present study utilizes a parasite pathogenic to man, thus providing information that may be particularly relevant to human filariasis. The assay system is readily maintainable since micro- filariae may be harvested many times from a single infected jird, while outbred CF1 mice are relatively inexpensive and sustain long-lasting micro- filaraemia. The need for the development of new therapeutic agents against filariasis has recently been stressed.d Since the development of diethylcarbamazine in 1947, very few new microfilaricidal drugs have been introduced or widely tested for the treatment of human infections and none has supplanted diethyl- carbamazine. This is due in part to the lack of suitable animal models. Although this drug has been shown to reduce the level of microfilaraemia in experimental animals such as cotton rats infected with L. carinii (3) and dogs with Dirofilaria immitis (4), few pharmacological studies of diethylcar- bamazine have employed animals infected with human filarial parasites (5). We found that doses that reduced microfilarial levels in humans with B. malayi infection (6 mg/kg for 10 days) (6) were effective in eliminating more than 90% of the circulating microfilariae in mice. Furthermore, over the range tested, the microfilaricidal effect was dose- dependent. The B. malayi murine model may there- fore be used to study the efficacy, dose-dependence, and mechanism of action of new microfilaricidal drugs, and to compare them in these respects with diethylcarbamazine. ACKNOWLEDGEMENTS The authors would like to acknowledge the assistance of Rebecca Davis in obtaining microfilariae from jirds and the Lederle Company, Pearl River, NY, USA, for supplying the diethylcarbamazine. REFERENCES 1. HAWKING, F. & SEWELL, P. British journal of phar- macology, 3: 285-296 (1948). 2. SASA, M. Human filariasis, Baltimore, University Park Press, 1976, pp. 646-647. 3. HEWrrr, R. L. ET AL. Journal of laboratory and clinical medicine, 32: 1293-1303 (1947). 4. SASA, M. Human filariasis, Baltimore, University Park Press, 1976, p. 744. 5. EDESON, J. F. B. & LAING, A. B. G. Annals of tropical medicine and parasitology, 53: 394-399 (1959). 6. TURNER, L. H. & SODHY, L. S. Annals of tropical medicine and parasitology, 53: 268-273 (1959). d Report of the first meeting of the Scientific Working Group on Filariasis. WHO unpublished document TDR/MPD/SWG-FIL (1)/ 77.3.