Establishment of a partly DFMO-sensitive primate model of Trypanosoma rhodesiense sleeping sickness

Acta Tropica, 59(1995)71-73 (c) 1995 Elsevier Science B.V. All rights reserved 0001-706X/95/$9.50

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ACTROP 00446

Short Communication

Establishment of a partly DFMO-sensitive primate model of Trypanosoma rhodesiense sleeping sickness E . M . E . B u r u d i * , S.M. K a r a n j a , A.I. N j u e , J.B. Githiori, J.M. Ndung'u Kenya Trypanosomiasis Research Institute (KETRI), P.O. Box 362, Kikuyu, Kenya Received 30 September 1994; revision received 28 November 1994; accepted 28 November 1994 Key words: Keywords: Trypanosoma brucei rhodesiense; D,L-~-Difluoromethylornithine; Vervet monkey; Sleeping sickness

Human African Trypanosomiasis (HAT) caused by T.b. rhodesiense occurs mainly in Eastern and Central Africa (Manson-Bahr and Bell, 1989). Unlike the West African form caused by T.b. gambiense that manifests as a chronic disease, the East African type is an acute complex of syndromes lasting less than a year. It has a three-phase course; first as the haemo-stage when parasites are in blood circulation with no central nervous system (CNS) invasion, then the second phase which is a transitional one when the parasites are present in the cerebrospinal fluid (CSF) with no CNS parenchymal infection and, thirdly the late stage, also called the meningoencephalitic phase, when the CNS parenchyma is invaded by parasites. For the management of the haemo-phase of the disease, suramin is the drug of choice for T.b. rhodesiense and T.b. gambiense infections whereas pentamidine may also be used in early T.b. gambiense infections. The late stage form is dependent on melarsoprol (Gutteridge, 1985). Treatment with melarsoprol is often associated with a potentially fatal and unpredictable encephalopathy in 5-10% of patients. D,L-~-Difluoromethylornithine (DFMO) is a promising antitrypanosomal compound for the treatment of late stage trypanosomiasis that is a selective enzymeactivated irreversible inhibitor of ornithine decarboxylase (Metcalf et al., 1978). This compound has been highly successful in the treatment of late stage T.b. gambiense infections (Nieuwenhove et al., 1985; Doua and Yapo, 1993), and also acts synergistically with several trypanocidal compounds against early and late stages of T.b. brucei infections (McCann et al., 1983). The use of DFMO in the field is hampered by its ineffectiveness in late stage T.b. rhodesiense infections, and the prohibitive cost of treatment of patients with late stage T.b. gambiense infections (Kuzoe, 1993). Studies using this compound in combination with other drugs in experimental animals have yielded very promising results (Clarkson et al., 1984; Jennings, 1993). *Corresponding author. Fax: +254 0154-32397. SSDI 0001-706X(94)00081-6

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Studies have been going on in our laboratory to establish a DFMO-sensitive vervet monkey (Cercopithecus aethiops) model of T.b. rhodesiense sleeping sickness that could provide an opportunity for improving the chemotherapy and management of the late stage form of the disease in humans. This would enable easier extrapolation of the findings to the human patient since the disease in this animal species is a close simulation of sleeping sickness in man. Earlier work in vitro and in a mouse model of sleeping sickness in our laboratory have shown that a number of T.b. rhodesiense isolates have varying degrees of sensitivity/resistance to DFMO. When infectivity studies were carried out in vervet monkeys using 2 of the isolates (KETRI 2545 and KETRI 2772) the animals developed a sub-acute disease syndrome that progressed to the late stage form 70 to 100 days after infection. In an attempt to establish a DFMO-sensitive vervet monkey model, isolate KETRI 2772 was used since it manifested a higher sensitivity to D F M O in mice than KETRI 2545. Two vervet monkeys were infected by intravenous injection with 1 x 104 trypanosomes and routinely monitored for parasitaemia, clinical, haematological and CSF changes. Infection was followed by an acute phase lasting up to 6 weeks. Treatment was initiated on day 42 following infection, a time when parasites were already present in CSF but may not have invaded the CNS parenchyma (transition phase). D F M O was administered per os via a drenching needle, at 200 mg/kg body weight every 6 h for 28 days. This is comparable to the dosage for human patients (800 mg/kg body weight p/o for 35-42 days; presently being tested). Parasitaemia cleared after only 3 days of treatment, and subsequently parasites disappeared from CSF and remained so until 10 days following termination of treatment. No significant drug-related side effects were observed during the course of treatment. Relapse parasitaemia and CSF parasites were detected after 2 weeks of termination of treatment. The relapsing cases were treated with a suramin/DFMO combination starting on day 124 post-infection (p.i). D F M O was administered as in the primary infection, and suramin was given at 20 mg/kg body weight intravenously on days 1, 3, 6 and 10 of D F M O treatment. Following initiation of treatment, parasitaemia disappeared within 3 days whereas the CSF became negative of parasites within a week. During a follow-up period of sixty days, the animals remained negative of parasites in both blood and CSF. They regained their body weights and packed cell volume, and the CSF white blood cell counts dropped gradually from levels as high as 250/p.1 to 0 within a month of termination of treatment. From the above, it would appear that D F M O has some efficacy against T.b. rhodesiense KETRI 2772, and that it crosses the blood brain barrier (BBB) to a significant degree. However, its efficacy on late stage disease is tremendously enhanced by suramin, a highly efficacious drug for the haemo-stage of sleeping sickness. This is in agreement with earlier findings by Clarkson et al. (1984) who demonstrated some efficacy using a similar combination in experimental late stage disease caused by T.b. brucei in mice. However, these findings are interesting, since suramin is hardly known to cross the BBB. Earlier studies have demonstrated cure of CSF-infected monkeys with suramin alone (Apted, 1970; unpublished data from our laboratory), suggesting that the drug may be crossing the BBB, although not at a high concentration. This may be providing sufficient drug levels in the CNS for the drug to act synergistically with D F M O (Bacchi et al., 1982; Clarkson et al., 1984). However, the mechanism of D F M O and suramin synergism is yet to be

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unraveled. At the same time, given that the combination treatment in this study was done on a relapse infection following DFMO treatment as opposed to a primary infection, the parasites' degree of resistance following the inevitable process of selection during the primary treatment could have been amplified. A higher degree of sensitivity would therefore be expected when a primary infection is subjected to a similar treatment. In light of the foregoing findings, studies are underway to determine the efficacy of the suramin/DFMO combination on a primary infection of a larger number of animals, and including relevant controls.

Acknowledgments These investigations received support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases and the USAID (Grant No. 615-0258-G-55-00-1070). The authors are grateful for the excellent technical assistance by staff of the primate unit in KETRI. The work is published with permission from the Director KETRI.

References Apted, F.I.C. (1970) Treatment of human trypanosomiasis. In: H.W. Mulligan (ed.), The African Trypanosomiases, Wiley Interscience, New York, pp. 684-710. Bacchi, C.J., Nathan, H.C., Humer, S.H., McCann, P.P. and Sjoerdsma, A. (1982) Novel combination therapy of experimental trypanosomiasis by using bleomycin and D,L