Herbicides to Curb Human Parasitic Infections: in Vitro and in Vivo
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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5657-5661, June 1993 Microbiology Herbicides to curb human parasitic infections: In vitro and in vivo effects of trifluralin on the trypanosomatid protozoans (Leishmani/Trypaosoma/microtubule/dinltrolanine) MARION MAN-YING CHAN*t, MAX GROGLI, CHIANN-CHYI CHEN*, E. JAY BIENEN§, AND DUNNE FONG* *Department of Biological Sciences and Bureau of Biological Research, Rutgers, The State University of New Jersey, Piscataway, NJ 08855-1059; tDivision of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington, DC 20307; and §Department of Medical and Molecular Parasitology, New York University School of Medicine, 550 First Avenue, New York, NY 10016 Communicated by William Trager, March 11, 1993 (receivedfor review December 21, 1992) ABSTRACT Leishmaniasis is a major tropical disease for cancer therapy and anthelmintic drugs, such as benzimid- which current chemotherapies, pentavalent antimonials, are azole, also target these structures (14). inadequate and cause severe side effects. It has been reported Trifluralin has been commercially available and widely that trifluralin, a microtubule-disrupting herbicide, is inhibi- used for weed control since the 1960s (15, 16). This herbicide tory toLeishmania amazonensis. In this study, the in vitro effect is well characterized, from toxicity to shelf-life, and is oftrifluralin on different species oftrypanosomatid protozoans inexpensive to manufacture. The selective effect oftrifluralin was determined. In addition to L. anazonensis, trifluralin is and its analog, oryzalin, on L. amazonensis has been noted effective against Leishmania major and Leishmanw tropica, in our previous publications (17-19). As reported in those which cause cutaneous infections, Leishmania donovani, which studies, trifluralin binds to Leishmania tubulin, inhibits pro- causes visceral disease, Leishmania panamensis, which may mastigote proliferation, decreases amastigote-to-promasti- cause mucocutaneous infection, and Trypanosoma brucei, an gote transformation, and reduces infectivity of amastigotes. important human and veterinary pathogen. Moreover, most Moreover, trifluralin does not bind to rat brain tubulin and is encouragingly, trifluralin is effective in vivo as a topical oint- nontoxic to murine macrophages and human monocytes. No ment against L. major and Leishmania mexicana murine cuta- significant inhibition of proliferation was observed at a con- neous leishmaniasis. Thus, trifluralin is a promising lead drug centration 20 times the effective dose for parasites. for several related, prevalent tropical diseases: leishmaniasis, In addition, it has also been reported that trifluralin has an trypanosomiasis of animals, and, possibly, African trypanoso- antimalarial effect. At 1-5 ,AM, it inhibits the growth of miasis in hunsm. Plasmodium falciparum in culture. When mature gameto- cytes were incubated with trifluralin, at concentration as low Leishmaniasis is a major tropical disease prevalent in 80 as 0.01 ,uM, complete inhibition of exflagellation was ob- countries. Approximately 12 million cases are estimated and served (20). Thus, trifluralin is a promising lead drug for 350 million people are at risk (1). The current FDA-approved several tropical diseases, including leishmaniasis, African therapy is Pentostam. This drug needs to be given intrave- trypanosomiasis, and malaria. nously, produces severe adverse effects, and requires hos- However, for many drugs-e.g., L-leucine methyl ester, pitalization for treatment, and failure of treatment is not difluoromethylornithine, and sinefungin, efficacy differs sig- uncommon. Pentostam-resistant leishmanias have been de- nificantly among species of Leishmania (21-23). In this tected and the compound has low activity against cutaneous report, we assess the in vitro efficacy oftrifluralin against the leishmania infections (2). More effective and less toxic ther- different species of trypanosomatids (24) and the in vivo apeutic drugs are urgently needed, especially for cutaneous efficacy of this herbicide against Leishmania in infected lesions that may not need systemic treatment. murine models. Microtubules of Leishmania are potential targets for che- motherapy. A characteristic feature of this protozoan para- MATERIALS AND METHODS site, as well as other members of the family Trypanosomat- idae, is the presence offunctionally distinct a- Parasites. The following species of parasites were used for microtubules; the in vitro assays: Leishmania major (MHOM/SU/73/ and ,-tubulins are their most abundant protein (3). Flagellar 5ASKH, WR661), Leishmania donovani (MHOM/IN/80/ microtubules are required for locomotion, subpellicular ones DD8, WR657), Leishmania panamensis (MHOM/CR/87/ for maintenance of cell shape, cytopharyngeal ones for WR746), and Trypanosoma brucei (TREU 667). L. mexicana endocytosis, and those ofthe nuclear spindle for cell division. (MNEO/US/90/WR972) and L. major (MHOM/SU/74/ Tubulin biosynthesis is also required during amastigote-to- WR779) were used for the in vivo studies. promastigote transformation, and tubulin gene expression is Efficacy Against Promastigotes. Promastigotes (1-2 x 105 regulated by transcriptional and post-transcriptional control per ml) were cultured at 27°C in liver infusion tryptose (LIT) (4-11). Moreover, the 13-tubulin amino acid sequence of medium (18, 25). Trifluralin (0.1 M) (Dow Elanco, Indianap- mammals differs from that of Leishmania amazonensis by olis) was dissolved in acetone, serially diluted in LIT medium 18% (previously referred to as Leishmania mexicana ama- with 0.1% acetone, and then added to the cultures. After zonensis; ref. 12), and the a- and (3-tubulin amino acid incubation for 5-6 days, the parasites were counted by sequences of mammals differ from those of Trypanosoma hemacytometer and the percent survival was calculated by rhodesiense by 15-16% (13), so tubulin proteins may have comparison to the untreated control. Percent survival = distinct structures that can be identified as drug targets. This number ofparasites in treated sample/number ofparasites in approach oftargeting the microtubules is not unprecedented; untreated control. The publication costs of this article were defrayed in part by page charge Abbreviations: LIT, liver infusion tryptose; DMSO, dimethyl sul- payment. This article must therefore be hereby marked "advertisement" foxide. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5657 5658 Microbiology: Chan et al. Proc. Natl. Acad. Sci. USA 90 (1993) 120 Efficacy on Infectivity. J774A.1 murine macrophages L. major L. tropica (American Type Culture Collection TIB 67) were cultured at 100 4_5 x 106 per ml in RPMI-1640 medium containing 10%o fetal 80 calf serum. The cells were infected with Leishmania promas- tigotes at a host:parasite ratio of 1:5-20, which produced 60 65-90%o infection. On the third day, the extracellular pro- 40 mastigotes were removed by aspiration and the infected 20 macrophages were used for drug testing. For L. major infection in which many parasites failed to enter the macro- 0 phages, the extracellular promastigotes were removed by L panamensis L.donovani 100 separation on a 45:90 Percoll step gradient. Trifluralin, dis- solved in the same medium with 0.1% acetone, was added. 80 After another 3-day period, the culture supematants were 60 removed, the macrophages were washed and stained with 40 Giemsa, and the % infected macrophages was determined for each sample. From this, the infective index (% infected 20 macrophages in treated cultures/% infected macrophages in 0 untreated cultures) was calculated. L. donovani cultures .1 1 10 .1 1 10 100 were incubated at 37°C; Leishmania tropica and L. major cultures were incubated at 33°C, all in incubators with 5% concentration (gM) CO2 (18, 19). Efficacy Against Trypanosomes. In vitro cultivated blood- FIG. 1. Trifluralin inhibited promastigote proliferation of differ- stream trypomastigotes and procyclic trypomastigotes of T. ent Leishmania species. Trifluralin was added to the cultures of promastigotes. After incubation for 5-6 days, the parasites were brucei brucei TREU 667 were used for these studies. The counted by hemacytometer and the % survival was calculated by bloodstream forms were axenically cultivated at 37°C and 5% comparison to the untreated control. CO2 according to the method of Hirumi and Hirumi (26) in modified by the use of 20% horse serum. The HMI-9 medium many reports of drug efficacy in the literature are based on procyclic forms were differentiated from the bloodstream defined medium; therefore, the effect of trifluralin in a forms in a semi-defined medium as previously described and defined medium was also determined. When serum-free and have been maintained in culture at 27°C for several months protein-free hybridoma medium (Hybri-max, Sigma) was (27). Trifluralin was dissolved in 0.5% dimethyl sulfoxide used, the amount of drug needed to inhibit proliferation was (DMSO) and added to the cultures at the indicated concen- not significantly different (data not shown). trations. The number of parasites was counted daily. For the intracellular stage, trifluralin at 15,uM reduced the In Vivo Studies. L. mexicana and L. major strains, which infective index of L. donovani, L. major, and L. tropica by had been standardized for drug testing at the Walter Reed >25% (Fig. 2). Since the herbicide needs to enter the cultured Army Institute for Research, were used. BALB/c mice, macrophages