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Combinations of DL-alpha-difluoromethylornithine (DFMO; eflornithine; Ornidyl) with either suramin or melarsen oxide were found to be effective against acute laboratory model infections with Trypanosoma brucei rhodesiense. We used clinical isolates known to be resistant to these drugs when used singly. An infection with a melarsen oxide-refractory isolate was cured by a combination of low-dose DFMO (0.5% in the drinking water) plus low-dose suramin (1 mg/kg of body weight given intraperitoneally). Another strain, moderately resistant to arsenical drugs, was cured with combinations of 4% DFMO with 5 mg of melarsen oxide per kg. Furthermore, a combination of DFMO (2% in the drinking water) and suramin (20 mg/kg) provided a 100% cure rate in a central nervous system model, although the same doses of these drugs used singly were completely ineffective. The synergism of DFMO and suramin against an acute infection was improved when suramin was given at the end of the DFMO administration. No adverse interactions were observed when high doses of DFMO combined with high doses of suramin were administered to uninfected mice. These results suggest that combinations of DFMO and suramin should be examined clinically for activity in arsenical-drug-refractory cases of East African sleeping sickness.

The compound 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL73811), a potent inhibitor of S-adenosylmethionine decarboxylase, was effective in mice against six of eight clinical isolates of Trypanosoma brucei rhodesiense, the causative agent of East African sleeping sickness. In combination with the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO; Ornidyl), MDL73811 acted synergistically to cure seven of eight infections. MDL73811 was effective when given singly at 50 to 100 mg/kg of body weight per day for 7 days (osmotic pumps). In combination with subcurative DFMO levels (0.25 to 1.0% in drinking water for 7 days), the curative MDL73811 dose could be lowered to 25 or 50 mg/kg, depending on the isolate. Oral administration of the MDL73811-DFMO combination was also effective in an acute infection and in a long-term central nervous system model of Trypansoma brucei brucei infection. These data indicate that MDL73811 may be effective therapeutically in drug-refractory and late-stage East African trypanosomiasis.

5'-Deoxy-5'-(methylthio)adenosine (MTA) is a by-product of polyamine metabolism and is phosphoryolytically cleaved to adenine and 5-deoxy-5-(methylthio)ribose-1-phosphate (MTR-1-P) by MTA phosphorylase. In eukaryotes, adenine is subsequently salvaged and converted to nucleotides, while MTR-1-P is converted to methionine. We examined 5'-deoxy-5'-substituted analogs of MTA for trypanocidal activity in vitro and in vivo. 5'-Deoxy-5'-(hydroxyethyl)thioadenosine (HETA) and its 5'-bromo,5'-chloro and 5'-fluoro derivatives were cleaved by extracts of the African trypanosome Trypanosoma brucei brucei (Km for MTA, 11.5 microM; Km for HETA, 13.2 microM) in a phosphate-dependent reaction. HETA and the three halo analogs were 50% inhibitory to growth at 0.5 to 5.0 microM in vitro. Inhibition of growth was reversible by exogenous methionine and 2-keto-4-methylthiobutyric acid, an intermediate in methionine synthesis from MTR-1-P. HETA was selected for further study in vivo. When administered by miniosmotic pump (25 to 150 mg/kg/day for 7 days) to mice infected with T. brucei brucei, HETA effected 70 to 90% cure rates. Results of this study indicate that these analogs of MTA are converted to trypanocidal MTR-1-P analogs and that this approach deserves further consideration in the development of novel chemotherapy of trypanosomiasis.

Clinical isolates of Trypanosoma brucei rhodesiense, which were resistant to arsenical drugs in murine infections, were examined for resistance in vitro. A rapid lysis assay was developed which was able to predict in vivo sensitivity to melarsoprol (Mel B, Arsobal) and melarsen oxide. The assay was based on the finding that long slender bloodforms of drug-sensitive isolates would lyse in the presence of arsenicals upon incubation in heat-inactivated fetal bovine serum. On the basis of plots of decrease in the absorbance of trypanosome suspensions vs time of incubation with drug, L50 values, reflecting the drug concentration necessary for lysis of 50% of the cells within 30 min. were calculated for five strains. These values ranged from less than 30 microM for arsenical-sensitive strains to greater than 75 microM in proven arsenic refractory isolates. Calcium was essential for lysis, and the presence of the Ca2+ chelator EGTA (10 mM) in serum delayed lysis of sensitive strains. Ca2+ channel antagonists (Verapamil, Diltiazem), however, did not enhance lysis of refractory isolates when used at 20 to 30 microM. Intracellular concentrations of reduced trypanothione, the apparent target of arsenicals, were similar for all isolates, approximately 1.02 +/- 0.28 nmol/10(8) cells, as detected by monobromobimane derivitization and HPLC analysis. Uptake of melarsen oxide was found to be reduced in arsenical refractory strains. Uptake was judged by reduction of free reduced trypanothione as a result of formation of the trypanothione-arsenic complex Mel T. Little change was found in arsenical-resistant strains, but sensitive strains had 50 to 70% reductions in trypanothione levels after incubation with a low (1 microM) level of melarsen oxide.

DL-alpha-Difluoromethylornithine is an enzyme-activated inhibitor of ornithine decarboxylase and an antagonist of polyamine metabolism that has been successful in clinical trials against West African sleeping sickness caused by Trypanosoma brucei gambiense. Its potential for use against the more virulent East African form of the disease, caused by T. brucei rhodesiense, is not certain. We examined 14 East African clinical isolates from the Kenya Trypanosomiasis Research Institute strain bank plus 2 established isolates for susceptibility to DL-alpha-difluoromethylornithine and to standard trypanocides. Seven of 16 strains were partially or totally refractory to DL-alpha-difluoromethylornithine in our test system. Four strains were also refractory to arsenical drugs, and five were refractory to diamidines. The results indicate that other novel agents or combinations of established agents may be needed for chemotherapy of East African disease.

The triazole nucleoside derivatives 1-(5'-O-sulfamoyl-beta-D-ribofuranosyl) [1,2,4]triazole-3-carboxamide (2), 1-(5'-O-sulfamoyl-beta-D-ribofuranosyl) [1,2,4]triazole-3-thiocarboxamide (3), and 1-(5'-O-sulfamoyl-beta-D-ribofuranosyl)-[1,2,4]triazole-3- carbonitrile (4) were synthesized. Suitably protected triazole nucleosides were converted to their corresponding 5'-sulfamoyl derivatives, which on subsequent deprotection gave the desired compounds in good yields. The structures of compounds 2-4 were confirmed by X-ray crystallographic analysis. All three compounds showed significant antiparasitic activity in vitro, while 2 showed significant activity in vivo against Leishmania donovani and Trypanosoma brucei.

A system of prescreens and screen has been developed to select chelators as potential drugs against Trypanosoma brucei brucei EATRO 110. The chelators tested were nearly all commercially available, low molecular, and having moderate to high affinity for Fe(III). We prescreened 70 compounds showing heme-sparing or inhibitory activity in a Crithidia fasciculata growth system having excess Fe and minimal hemin. Of these, 45 were highly trypanocidal for suspensions of bloodstream T. b. brucei; criteria of activity here were immobilization, lysis, and loss of infectivity. Eighteen of the chelators highly active in the suspension prescreen were tried in T. b. brucei-infected mice. Thirteen of these chelators were curative in mice with 24-h infections, that is, they allowed survival greater than 30 days beyond the untreated controls. 3,4-Dihydroxycinnamic acid (caffeic acid), 2,9-dimethyl-1, 10 phenanthroline (neocuproine), and 2-pyridinecarboxaldehyde-2-pyridyl-hydrazone cured five out of five mice after an i.v dose of 100 mg/kg. Salicylaldehyde thiosemicarbazone cured five out of five mice at an i.p. dose of 500 mg/kg. Lesser activity was shown by several other chelators.

Two ornithine decarboxylase inhibitors, DL-alpha-difluoromethylornithine (eflornithine; DFMO) and a-monofluoromethyldehydroornithine methyl ester (delta MFMO X CH3) were compared in their ability to cure two distinct Trypanosoma brucei brucei central nervous system murine model infections. Both inhibitors cured the TREU 667 and LUMP 1001 isolates if used in combination with a single (20 mg/kg) injection of suramin, a trypanocide in current clinical use. The curative dose of delta MFMO X CH3 in combination with suramin was 1.09 g/kg/day, administered in the drinking water for 14 days; used with suramin, the curative dose of DFMO was 5.3 g/kg/day for 14 days (5 times the delta MFMO X CH3 dose required). In host animals, delta MFMO X CH3 was not toxic and was accumulated by trypanosomes 6-8 times faster than DFMO. Since DFMO by itself has been highly effective against T. b. gambiense infections in humans (12-15 g/day for 6 weeks) the present data suggest that delta MFMO X CH3 might be effective in a shorter regimen and at lower doses.

The EATRO 110 isolate of Trypanosoma brucei brucei was grown in rats for 60 h and the animals treated with the ornithine decarboxylase inhibitor alpha-DL-difluoromethylornithine 12 h or 36 h prior to sacrifice. Control untreated animals died 72-80 h after infection. Treated parasites were shorter and broader than the predominantly long slender forms found in untreated controls and many had two or more nuclei and kinetoplasts. Trypanosomes were purified from blood and examined for disruption of polyamine metabolism. ODC activity decreased by more than 99% after 12 h treatment and putrescine and spermidine levels also decreased dramatically. Spermine, not normally present in control cells, increased to detectable, low levels (less than 1 nmol mg-1 protein) after 36 h treatment. alpha-DL-Difluoromethylornithine-treated cells were unable to synthesize putrescine from [3H]ornithine but were able to convert [3H]putrescine + methionine to spermidine. 12-h treated parasites responded to polyamine depletion by assimilating radiolabeled polyamines in vitro at 2- to 4-times the rate of untreated cells. The metabolism of S-adenosylmethionine was also altered in treated parasites: decarboxylated S-adenosylmethionine increased more than 1000-fold over untreated cells while S-adenosylmethionine decarboxylase activity, associated with the formation of spermidine and spermine in other eukaryotes, paradoxically declined in treated cells. Synthesis of macromolecules was perturbed in treated parasites: rates of DNA and RNA synthesis declined 50-100%, while protein synthesis increased up to 4-fold in 36-h treated cells. alpha-DL-Difluoromethylornithine treatment progressively limits the parasites' ability to synthesize nucleic acids and blocks cytokinesis while inducing morphological changes resembling long slender leads to short stumpy transformation.

Kinetoplastid hemoflagellates are sensitive to growth inhibition by various purine analogs. In this study the activities of 9-deazainosine (9-DINO), formycin B, and sinefungin were compared in experimental murine Trypanosoma brucei subsp. brucei infections, both singly and in combination with the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO, eflornithine). Used singly, all of the purine analogs were able to suppress an acute T. brucei subsp. brucei infection. 9-DINO and formycin B were the most active. None of the purine analogs was curative when used singly against a strain causing chronic central nervous system infection. 9-DINO was highly effective when used in combination with DFMO in curing this central nervous system infection and another more stringent experimental infection. Neither sinefungin nor formycin B was active in combination with DFMO in curing the central nervous system experimental infection. 9-DINO was metabolized to phosphorylated derivatives of 9-deazaadenosine and 9-deazaguanosine by bloodstream trypomastigotes, but not by murine erythrocyte suspensions or kidney or liver homogenates--a potential rationale for the selectivity of the analog. These studies indicate that 9-DINO is a potent, nontoxic purine analog which, in combination with DFMO, is capable of late-stage cures of African trypanosomiasis.

Polyamines were determined by n-butanol extraction and thin-layer chromatography in four trypanosomatids: Trypanosoma brucei (rat infection) and cultures of Crithidia fasciculata, Leptomonas sp., and Trypanosoma mega. All had putrescine and spermidine but no detectable spermine. Putrescine and spermidine levels were quantitated for extracts of leptomonas during the normal growth cycle. Spermidine values peaked 18 h before peak cell populations. Spermidine-putrescine ratios for all organisms were related to the presumed phylogeny of the group.

alpha-Difluoromethylornithine (RMI 71,782), a specific irreversible inhibitor of the first step in polyamine biosynthesis, that is, the formation of putrescine from ornithine by ornithine decarboxylase, cures mice infected with a virulent, rodent-passaged strain of Trypanosoma brucei brucei. This parasite is closely related to the trypanosomes that cause human sleeping sickness. The drug, which is remarkably nontoxic, was effective when administered in drinking water or by intubation. The ability of the compound to inhibit ornithine decarboxylase in vitro was demonstrated by the reduced amounts of putrescine synthesized from tritiated ornithine in Trypanosoma brucei suspensions. These observations direct attention to polyamine metabolism as a target for chemotherapy of parasitic diseases.

DL-alpha-Difluoromethylornithine, a polyamine biosynthesis inhibitor, and bleomycin, a currently used antineoplastic agent, have each previously been shown to be curative for acute short-term infections of mice with Trypanosoma brucei brucei, an African trypanosome closely related to those that cause the human disease African sleeping sickness. These agents were tested singly and in combination in a previously described mouse model of sleeping sickness with demonstrable brain involvement. The original model is extended by using two additional strains of outbred mice and by demonstrating that melarsoprol, an arsenical and currently the only drug used for human African trypanosomiasis involving the brain, was also curative for these brain infections. Neither difluoromethylornithine nor bleomycin alone was curative for the brain infections; however, many combinations of the two drugs were found to be 100% curative with no evidence of immediate toxicity.

A series of phthalanilides and related compounds were tested on a short-term, fulminating, mouse infection of Trypanosoma brucei brucei (EATRO 110 isolate). The most effective compound was [4,4'-bis (4-methylimidazolin-2-yl)-terephthalanilide] which had a cure rate of 75% at 0.1 mg/kg body weight and 100% at 0.5 mg/kg when administered as three single daily intraperitoneal injections starting 24 hours post-infection. Several related phthalanilides and similarly substituted ureas showed definite but lower activity. In tests with a chronic neurotropic T. b. brucei isolate (TREU 667), cure rates greater than 90% were obtained with 10 or 25 mg/kg [4,4'-bis(4-methylimidazolin-2-yl)-terephthalanilide]. Cured animals survived for at least 200 days after infection with no evidence of recrudescence of parasitemia or of toxicity; blood or brain homogenates of cured animals were non-infective to immunosuppressed animals. These studies indicate that this series of compounds, previously studied as antitumor agents, should be re-examined as potential trypanocides.

DL-alpha-Difluoromethylornithine (DFMO), a specific enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC) was previously shown to cure mice infected with Trypanosoma brucei brucei, a parasite of game and cattle in Africa and Trypanosoma brucei rhodesiense, a human African Sleeping Sickness pathogen. Our studies now indicate that DFMO blocks ornithine decarboxylase and lowers trypanosome polyamine levels in vivo. Polyamine uptake in T.b. brucei also resembles that previously described for mammalian cells. The therapeutic potential of DFMO can now also be extended to another human pathogen, Trypanosoma brucei gambiense. Finally, DFMO acts synergistically with another drug, bleomycin, to cure acute trypanosome infections, and furthermore, this same drug combination provides a new approach to the treatment of trypanosomal infections of the central nervous system.

Although bleomycin is a known pulmonary toxin, results presented herein indicate its relative safety for treatment of trypanosomiasis. More than 4 times the curative dose in this acute model of infection does not induce significant alterations of lung hydroxyproline levels, which are known to directly correlate with histopathological criteria of pulmonary fibrosis.

The antitumour antibiotic bleomycin, supplied as commercial BlenoxaneR (a mixture of bleomycinic acids), at 7 or 14 mg/kg prolonged life greater than 30 days beyond death of controls without relapse or sign of trypanosomes in the peripheral blood of mice infected with Trypanosoma b. brucei EATRO 110. Control mice died in three to four days. The purified A2 and B2 bleomycin congeners were also active at this dose. Spermidine, spermine and hirudonine (1,8-diamidinospermidine) but not putrescine, co-administered with drug, annulled the curative effects of these compounds, as signalled by appearance of trypanosomes in the blood and death of the animals.

The babesicides imidocarb and amicarbalide, which have structural similarities to the antitrypanosomatid diamidines, proved active against Trypanosoma brucei mouse infections: both cured infections when doses were administered daily for 3 days 24 h post-inoculation (curative dose imidocarb, 10 mg/kg; amicarbalide, 25 mg/kg). Mice were considered cured after survival 30 days longer than untreated infected controls, with no trypanosomes present in blood or cerebrospinal fluid smears. Both agents also cured when administered 48 and 72 h after challenge with T. brucei and prolonged the lives of animals 94 h after challenge. The results are discussed in respect to the potential of these carbanilides and their precursors, the antitumor phthalanilides, as lead compounds in chemotherapy of mammalian trypanosomiases.

Using a previously described mouse model of late-stage African trypanosomiasis (i.e., involvement of the central nervous system), we demonstrate that a combination of DL-alpha-difluoromethylornithine (DFMO) and suramin is curative. In the curative protocol, DFMO is given as a 2% solution in the drinking water for 14 days and suramin is administered as a single dose (20 mg/kg intravenously) on day 1 of DFMO administration. Since: 1) DFMO has very low toxicity, 2) suramin is one of the least toxic of the presently used trypanocides, and 3) suramin and DFMO act synergistically in mouse models of both acute and late stage trypanosomiasis, we conclude that this combination offers special promise in the treatment of African trypanosomiasis in man.

(Diacetyldiphenylurea)bis(guanylhydrazone) (DDUG) functions as a cationic trypanocide antagonized in vivo by exogenous concomitant addition of the biologically active polyamine, spermine. It also inhibits the DNA polymerases of L1210 murine leukemia cells. We have found that DDUG stimulates Rauscher murine leukemia virus DNA polymerase activity in a manner similar to polyamines. Such stimulation does not occur if DNA synthesis is carried out on spermine + activated DNA complexes. We also show that the in vivo antileukemic activity of DDUG in the L1210 ascites mouse model is antagonized by biologically active polyamines. These studies suggest a new intracellular target for the antileukemic activity of DDUG: interference with polyamine function.