In Vitro and In Vivo Activities of E5700 and ER-119884, Two Novel Orally Active Squalene Synthase Inhibitors, against Trypanosoma cruzi

Chagas' disease is a serious public health problem in LatinAmerica, and no treatment is available for the prevalent chronicstage. Its causative agent, Trypanosoma cruzi, requires specificendogenous sterols for survival, and we have recently demonstratedthat squalene synthase (SQS) is a promising target for antiparasiticchemotherapy. E5700 and ER-119884 are quinuclidine-based inhibitorsof mammalian SQS that are currently in development as cholesterol-and triglyceride-lowering agents in humans. These compoundswere found to be potent noncompetitive or mixed-type inhibitorsof T. cruzi SQS with K_i values in the low nanomolar to subnanomolarrange in the absence or presence of 20 µM inorganic pyrophosphate.The antiproliferative 50% inhibitory concentrations of the compoundsagainst extracellular epimastigotes and intracellular amastigoteswere ca. 10 nM and 0.4 to 1.6 nM, respectively, with no effectson host cells. When treated with these compounds at the MIC,all of the parasite's sterols disappeared from the parasitecells. In vivo studies indicated that E5700 was able to providefull protection against death and completely arrested the developmentof parasitemia when given at a concentration of 50 mg/kg ofbody weight/day for 30 days, while ER-119884 provided only partialprotection. This is the first report of an orally active SQSinhibitor that is capable of providing complete protection againstfulminant, acute Chagas' disease.

INTRODUCTION

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Introduction

Chagas' disease, a parasitic disease caused by the kinetoplastidprotozoan Trypanosoma (Schizotrypanum) cruzi, is still one ofthe most serious public health problems in Latin America, despiterecent advances in the control of the vectorial and transfusionaltransmission of the parasite (36). There are ca. 18 millionpeople currently infected with T. cruzi, most of them in thechronic phase, which can lead to severe heart and gastrointestinallesions (36). Current chemotherapy, based on the nitrofurannifurtimox (Bayer) and the nitroimidazole benznidazole (Roche),is unsatisfactory as these compounds are effective only forrecent infections and have frequent toxic side effects (21,24, 28). Studies have shown that T. cruzi has a strict requirementfor specific endogenous sterols (ergosterol and analogs) forsurvival and growth and cannot use the abundant supply of cholesterolpresent in its mammalian host (21-23, 25). Ergosterol biosynthesisinhibitors with potent in vitro activity and special pharmacokineticproperties in mammals (large volumes of distribution and longhalf-lives) can induce the radical cure of parasitic infectionin experimental animal models of both acute and chronic Chagas'disease (21-23, 25). Squalene synthase (SQS; EC 2.5.1.21) catalyzesa head-to-head reductive dimerization of two molecules of farnesylpyrophosphate (FPP) in a two-step reaction to form squalene(Fig. 1) (14), this reaction being the first committed stepin sterol biosynthesis. This enzyme is currently under intensestudy as a possible target for cholesterol-lowering agents inhumans (1, 12, 35), but it has been shown only recently as apromising target for antiparasitic chemotherapy (26, 27). Forthe present work, we investigated the in vitro and in vivo activitiesof E5700 and ER-119884, two novel quinuclidine SQS inhibitorscurrently in development by Eisai Company, Ltd. (Ibaraki, Japan)as cholesterol- and triglyceride-lowering agents in humans (T.Okada, D. Shinmyo, K. Tanaka, N. Kurusu, K. Miyazaki, H. Sugumi,H. Ikuta, M. Ito, M. Yanagimachi, H. Hiyoshi, T. Saeki, andS. Abe, Abstr. 226th Am. Chem. Soc. Natl. Meet., abstr. MEDI-293,2003; M. Yanagimachi, H. Hiyoshi, M. Ito, N. Yasuda, T. Okada,H. Ikuta, and T. Saeki, Abstr. 13th Int. Symp. Atherosclerosis,Atherosclerosis Suppl. 4, p. 226, 2003) against T. cruzi andfound that they also have potent and specific antiparasiticactivity.

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FIG. 1. Chemical reaction catalyzed by SQS.

(A short version of this work has been presented previously[J. A. Urbina, J. L. Concepcion, G. Visbal, S. Rangel, R. Lira,and A. Caldera, Abstr. 41st Intersci. Conf. Antimicrob. AgentsChemother., abstr. 493, 2001].)

MATERIALS AND METHODS

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Materials and Methods

Parasite. The EP (5) and Y stocks of T. cruzi were used in this study.The handling of live T. cruzi organisms was done according toestablished guidelines (7).

SQS purification and assay. SQS activity was assayed using highly purified T. cruzi glycosomesand microsomes, which were isolated as described previously(27), as enzyme sources. The assay was carried out by the spot-washmethod originally developed by Tait (18).

Studies of in vitro antiproliferative activity. The epimastigote form of the parasite was cultivated in liverinfusion tryptose medium (5) supplemented with 10% newborn calfserum (Gibco) at 28°C with strong agitation (120 rpm). Thecultures were initiated with a cell density of 2 x 10⁶ epimastigotesper ml, and the drug was added when the cell density was 0.5x 10⁷ to 1 x 10⁷ epimastigotes per ml. Cell densities were measuredwith an electronic particle counter (model ZBI; Coulter ElectronicsInc., Hielah, Fla.) and by direct counting with a hemocytometer.Cell viability was monitored by trypan blue exclusion analysisusing light microscopy. Amastigotes were cultured in Vero cellsmaintained in minimal essential medium supplemented with 1%fetal calf serum in a humidified 95% air-5% CO₂ atmosphere at37°C as previously described (30-33). Briefly, the cellswere infected with 10 tissue culture-derived trypomastigotesper cell for 2 h and then washed three times with phosphate-bufferedsaline to remove nonadherent parasites; fresh medium with andwithout drugs was added, and the cells were incubated for 96h with a medium change at 48 h. Quantification of the infectedcells and the parasites per cell was done by use of light microscopy,and statistical analysis of the results was carried out as describedpreviously (30-33).

Studies of lipid composition. For the analysis of the effects of drugs on the lipid compositionof the epimastigotes, total lipids from control and drug-treatedcells were extracted and fractionated into neutral and polarlipid fractions by silicic acid column chromatography and gas-liquidchromatography (32, 33). The neutral lipid fractions were firstanalyzed by thin-layer chromatography (on Merck 5721 silicagel plates with heptane-isopropyl ether-glacial acetic acid[60:40:4] as the developing solvent) and conventional gas-liquidchromatography (isothermal separation in a 4-m glass columnpacked with 3% OV-1 on Chromosorb 100/200 mesh with nitrogenas the carrier gas at a flow rate of 24 ml/min and flame ionizationdetection in a Varian 3700 gas chromatograph). For quantitativeanalysis and structural assignments, the neutral lipids wereseparated in a capillary high-resolution column (25-m by 0.20-mm[inside diameter] Ultra-2 column, 5% phenyl-methyl-siloxane,0.33-µm film thickness) in a Hewlett-Packard 6890 Plusgas chromatograph equipped with an HP5973A mass-sensitive detector.The lipids were dissolved in chloroform and injected; the columnwas kept at 50°C for 1 min, and then the temperature wasincreased to 270°C at a rate of 25°C min^–1 andfinally to 300°C at a rate of 1°C min^–1. The carriergas (He) flow was kept constant at 0.5 ml min^–1. The injectortemperature was 250°C, and the detector was kept at 280°C.

In vivo studies. The in vivo studies were carried out using a murine model ofacute Chagas' disease as previously described (30-33). Briefly,groups of 10 female NMRI-IVIC (20- to 25-g) mice were infectedwith 10⁵ T. cruzi bloodstream trypomastigotes per mouse, andoral treatment with E5700 or ER-119884 at a concentration of25 or 50 mg/kg of body weight/day was started 24 h later andwas given for 30 consecutive days. Negative (untreated) controlsreceived only the vehicle, 1% methylcellulose plus 0.5% Tween80. As positive controls, a group of animals received the referencedrug nifurtimox at 50 mg/kg/day. The animals were monitoredfor 40 days postinoculation (p.i.). The survival rate was monitoreddaily, and parasitemia was checked weekly by direct microscopicexamination as described previously (30-33).

Statistical analysis. The Kaplan-Meier nonparametric method was used to estimate thesurvival functions of the different experimental groups. Ranktests (log rank, which gives equal weight to all observations,and the Peto-Peto-Wilcoxon test, which uses an estimate of thesurvival functions for its weightings) were used to comparethem. The analyses were done with the Survival Tools packagefor StatView version 4.5 (Abacus Concepts Inc., Berkeley, Calif.)run on a Power Macintosh G4 Cube computer.

Drugs. E5700 {(3R)-3-[[2-benzyl-6-[(3R,4S)-3-hydroxy-4-methoxypyrrolidin-1-yl]pyridin-3-yl]ethynyl]quinuclidin-3-olmonohydrate} (Fig. 2, top) and ER-119884 {(3R)-3-[[2-benzyl-6-(3-methoxypropyloxy)pyridin-3-yl]ethynyl]quinuclidin-3-ol}(Fig. 2, bottom) were provided by Tsukuba Research Laboratories,Eisai Co. For in vitro studies the compounds were dissolvedin dimethyl sulfoxide and added directly to the growth medium;the final concentration of dimethyl sulfoxide in the mediumnever exceeded 1% (vol/vol) and had no effect on the proliferationof extracellular or intracellular parasites.

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FIG. 2. Chemical structures of E5700 and ER-119884.

RESULTS

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Results

Inhibitory activities against purified T. cruzi SQS. SQS has a dual subcellular localization in T. cruzi, being associatedwith both glycosomes (peroxisome-like organelles typical oftrypanosomatid parasites) and microsomes (27). E5700 and ER-119884were extremely potent inhibitors of both forms of the enzyme,with 50% inhibitory concentrations (IC₅₀s) (at saturating concentrationsof the enzyme substrates FPP and NADPH) in the low nanomolarto subnanomolar range in the absence or presence of 20 µMinorganic pyrophosphate (PP_i) (Table 1). The compounds wereshown to be noncompetitive (E5700) (Fig. 3) or mixed-type (ER-119884)(Fig. 4) inhibitors, with K_i values mostly in the subnanomolarrange (Table 2).

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TABLE 1. Inhibitory activities of E5700 and ER-119884 against SQS from T. cruzi epimastigotes^a

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FIG. 3. Kinetics of T. cruzi glycosomal SQS inhibition by E5700. Double reciprocal plots of the enzyme activity as a function of various concentrations of FPP, in the presence of the indicated fixed concentrations of E5700 in the absence (A) and presence (B) of 20 µM PP_i, are presented. Insets, secondary plots of the intercepts as functions of E5700. SQS activity was measured in the presence of 0.1% Triton X-100. 1/V, inverse of the reaction rate; 1/Vm app, inverse of the maximum apparent reaction rate; p, protein content of the sample.

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FIG. 4. Kinetics of T. cruzi glycosomal SQS inhibition by ER-119884. Double reciprocal plots of the enzyme activity as a function of various concentrations of FPP, in the presence of the indicated fixed concentrations of ER-119884 in the absence (A) and presence (B) of 20 µM PP_i, are presented. Insets, secondary plots of the slopes as functions of E5700. SQS activity was measured in the presence of 0.1% Triton X-100. 1/V, inverse of the reaction rate; 1/Vm app, inverse of the maximum apparent reaction rate; p, protein content of the sample.

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TABLE 2. Inhibition kinetic constants for E5700 and ER-119884 against SQS from T. cruzi epimastigotes^a

In vitro antiproliferative activities. Both quinuclidine derivatives were potent growth inhibitorsagainst the extracellular epimastigote form of the parasite,with an IC₅₀ and a MIC (defined as the minimal concentrationrequired to induce complete growth arrest) of 8 and 30 nM, respectively,for E5700 and 11 and 100 nM, respectively, for ER-119884 (Fig.5). Growth arrest and cell lysis induced by both compounds wereassociated with the disappearance of the parasite's endogenoussterols, no accumulation of lanosterol or squalene, and a concomitantincrease in the content of externally derived cholesterol (5)(Tables 3 and 4). Against the clinically relevant intracellularamastigote form, the compounds were even more active: the IC₅₀and the minimum concentration required to reduce the intracellularparasite load by >99% (MICa) were 1.7 and 100 nM, respectively,for E5700 and 0.38 and 30 nM, respectively, for ER-119884 (Fig.6). No deleterious effects of any of the compounds against hostcells were observed at concentrations up to 10-fold higher thanthe MIC against amastigotes, indicating a selective antiparasiticeffect.

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FIG. 5. Effects of E5700 (A) and ER-119884 (B) on the proliferation of T. cruzi epimastigotes. Epimastigotes were cultured in liver infusion-tryptose medium at 28°C, with agitation, as described in Materials and Methods. Arrows indicate the times of addition of the drug at the indicated concentrations. Experiments were carried out in triplicate, and each error bar represents 1 standard deviation.

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TABLE 3. Free sterols and precursors present in T. cruzi epimastigotes (EP stock) grown in the presence or absence of E5700^a

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TABLE 4. Free sterols and precursors present in T. cruzi epimastigotes (EP stock) grown in the presence or absence of ER-119884^a

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FIG. 6. Concentration dependence of the effects of E5700 (A) and ER-119884 (B) on the proliferation of T. cruzi amastigotes cultured in Vero cells at 37°C. The percentage of infected cells, the number of amastigotes per cell, and the number of Vero cells per field after 96 h of incubation are shown as functions of the drug concentration. Vero cells were infected with T. cruzi as described in Materials and Methods. Experiments were carried out in quadruplicate, and each error bar represents 1 standard deviation.

In vivo antiparasitic activities. Using a murine model of acute Chagas' disease, in which animalswere infected with 10⁵ T. cruzi bloodstream trypomastigotesper mouse and oral treatment was started 24 h p.i., it was foundthat E5700 given for 30 days had a dose-dependent effect onparasitemia and survival and at 50 mg/kg/day suppressed developmentof parasitemia (Fig. 7) and provided full protection againstdeath, while all of the control (untreated) animals were dead35 days p.i. (Fig. 8). The results were comparable with thoseobtained with the reference drug, nifurtimox, also given at50 mg/kg/day. In contrast, ER-119884 was able to induce only50% survival at 50 mg/kg/day, while no effects on parasitemiaor survival were seen when it was given at 25 mg/kg/day (Fig.6 and 7). However, even in animals treated with 50 mg of E5700/kg/dayfor 30 days, no cures of the parasitic infections were obtained,as indicated by the presence of low but detectable levels ofcirculating parasites after the discontinuation of treatment.No discernible toxic side effects to the mice were associatedwith any of the quinuclidine derivatives.

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FIG. 7. Effects of E5700, ER-119884, and nifurtimox on parasitemia in a murine model of acute Chagas' disease. NMRI-IVIC mice were inoculated with 10⁵ blood trypomastigotes of the Y strain, and oral treatment was started 24 h later. All drugs were given orally at the indicated doses for 30 consecutive days. For details, see Materials and Methods.

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FIG. 8. Effects of E5700, ER-119884, and nifurtimox on survival in a murine model of acute Chagas' disease. NMRI-IVIC mice were inoculated with 10⁵ blood trypomastigotes of the Y strain, and oral treatment was started 24 h later. All drugs were given orally at the indicated doses for 30 consecutive days. Statistical analysis using the log rank (Mantel-Cox) test indicated very significant (P < 0.0001) differences in survival rates between the control (untreated) animals and those that received E5700 or nifurtimox at 50 mg/kg/day and significant (P = 0.04) differences between the controls and those receiving E5700 at 25 mg/kg/day or ER-119884 at 50 mg/kg/day but no difference (P > 0.4) between the controls and those treated with ER-119884 at 25 mg/kg/day. For details, see Materials and Methods.

DISCUSSION

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Discussion

Quinuclidine derivatives are well-known inhibitors of mammalianSQS (4, 8, 9, 11, 19, 20, 34), and it has been shown previouslythat the prototype compound BPQ-OH [3-(biphenyl-4-yl)-3-hydroxyquinuclidine](4, 11) is also a potent inhibitor of T. cruzi SQS and has selectiveanti-T. cruzi and anti-Leishmania activity in vitro (27). Thenovel quinuclidine derivatives studied in the present work,E5700 and ER-119884 (Fig. 2), were 1 order of magnitude morepotent than BPQ-OH as inhibitors of the parasite enzyme, andtheir activity was further enhanced by the presence of micromolarlevels of PP_i (Fig. 3 and 4 and Tables 1 and 2). The compoundswere noncompetitive or mixed-type inhibitors, and their K_i values(Table 2) were 3 to 4 orders of magnitude lower than the K_mvalues for the substrate FPP and 4 to 5 orders of magnitudelower than the corresponding values for NADPH (27). Taken together,these facts strongly support the notion that quinuclidine derivatives,with positively charged aza groups in the centers of relativelyhydrophobic molecules (Fig. 2), could act by mimicking the carbocationictransition state of the reaction catalyzed by SQS (Fig. 1),which also involves an ion pair with the leaving pyrophosphategroup (2, 10, 14, 15). In this context it is interesting thatwe have recently discovered that T. cruzi and other trypanosomatidand apicomplexan parasites contain high levels of PP_i and othershort-chain polyphosphates that are distributed throughout thecells, although they are mostly stored in specialized organellescalled acidocalcisomes (13, 16, 29). It is an intriguing possibilitythat the presence of significant levels of PP_i in the intracellularmilieu of these parasites may make them more susceptible thanother eukaryotes to the effects of quinuclidine derivativeson SQS, including that in their mammalian host cells, wherePP_i levels are kept very low due to the presence of highly activesoluble pyrophosphatases.

Both quinuclidine derivatives were potent growth inhibitorsin vitro, particularly against the clinically relevant formsof the parasite, the intracellular amastigotes, with IC₅₀s inthe low nanomolar to subnanomolar range (Fig. 6); they are amongthe most active anti-T. cruzi compounds ever tested (21, 24,28). In epimastigotes, the growth arrest and loss of cell viabilityinduced by these compounds were associated with an almost completedisappearance of the parasite's endogenous sterols (Fig. 5 andTables 3 and 4), as seen previously with other SQS inhibitorsacting against this parasite (26, 27). This altered chemicalcomposition should lead to drastic modifications of the physicalproperties of the cell's membranes, such as passive permeabilityand sterol-mediated lipid-protein interactions in lipid rafts(3, 17). In contrast, there were no effects on the viabilityand proliferation of the mammalian host cells, even at concentrationsmore than 10 times higher than those required to eradicate theintracellular parasites (Fig. 6), despite the fact that thesecompounds are also very potent inhibitors of mammalian SQS (Okadaet al., Abstr. 226th Am. Chem. Soc. Natl. Meet., abstr. MEDI-293).As discussed elsewhere (27), the selective antiparasitic activityof these compounds is probably explained by the capacity ofthe host cells to compensate for the blockade of de novo cholesterolsynthesis by up-regulating the expression low-density lipoproteinreceptors and taking this sterol from the growth medium (6).In contrast, there is no way for the parasite to compensatein this manner for the quinuclidine-induced blockade of ergosterolbiosynthesis, as there are no appreciable amounts of ergosterolin the host cells or growth medium. It is also possible, asdiscussed above, that whole mammalian cells are intrinsicallyless susceptible to SQS inhibition by quinuclidine compounds,due to the absence of intracellular PP_i in them. In this context,it is noteworthy that E5700, in contrast to its potent anti-T.cruzi action, has very low in vitro antifungal activity (MIC,>200 µg/ml or 440 µM for Candida albicans, Aspergillusfumigatus, and Saccharomyces cerevisiae) [Eisai Co., unpublisheddata], a fact most probably explained by poor penetration bythe compound due to the presence of cell walls.

Although the two quinuclidine derivatives had comparable invitro anti-T. cruzi activities, E5700 was clearly more potentin vivo, as it was able to completely suppress the developmentof parasitemia and provide full protection against death whengiven at 50 mg/kg/day, while ER-119884 at the same dose providedonly partial protection, comparable to that seen with 25 mgof E5700/kg/day (Fig. 7 and 8). When the effects of these compoundson cholesterol and triglyceride synthesis and on plasma lipidlevels were investigated, the same patterns of in vitro andin vivo activities emerged: the compounds were of comparablepotencies against purified rat SQS and in reducing cholesteroland triglyceride synthesis in primary cultured rat hepatocytes,but E5700 was much more active in reducing plasma lipid levelsin rhesus monkeys after a 7-day course of treatment (Okada etal., Abstr. 226th Am. Chem. Soc. Natl. Meet., abstr. MEDI-293;Yanagimachi et al., Abstr. 13th Int. Symp. Atherosclerosis,Atherosclerosis Suppl. 4, p. 226). The higher in vivo activityof E5700 as a cholesterol-lowering and antiparasitic agent ismost probably due to better pharmacokinetic properties. Studiesof rats (Eisai Co., unpublished data) have shown that ER-119884has a clearance rate threefold higher than that of E5700 afterintravenous injection at 3 mg/kg and is associated with four-to sixfold-lower maximum concentrations in plasma and five-to sixfold-lower values for the area under the plasma concentration-timecurve from 0 h to infinity after oral doses of 3 to 10 mg/kgare given; a similar pattern was observed for rhesus monkeys.Studies of mice (Eisai Co., unpublished data) found that themaximum concentrations of E5700 in plasma attained after oraldoses of 30 to 100 mg/kg/day were 10- to 70-fold higher thanthe MIC for intracellular T. cruzi amastigotes determined inthe present work (MICa, 100 nM) (Fig. 6); furthermore, in therat model, this compound was found to have a wide tissue distribution,attaining levels in the tissue of relevant organs such as heart,spleen, liver, and small and large intestines that were 1 to2 orders of magnitude higher than those found in plasma (EisaiCo., unpublished data). Thus, assuming a similar distributionin mice, intracellular amastigotes in animals treated with 50mg of E5700/kg/day are expected to be exposed to steady-statedrug levels that are 2 to 3 orders of magnitude higher thanthe MICa, a fact which could explain the complete blockade ofparasite proliferation induced by this compound. Nevertheless,no cures of parasitic infection were obtained in these animalseven after 30 days of treatment, a fact probably related tothe short mean residence time (MRT = AUMC/AUC, where AUMC isthe area under the first moment of the plasma concentration-timecurve and AUC is the area under the curve) of E5700 in mice(2.5 to 3.8 h [Eisai Co., unpublished data]). New experimentsexploring the effects of more frequent dosing as well as combinationswith other sterol biosynthesis inhibitors such as antifungalazoles (inhibitors of sterol C14- ${alpha}$ -demethylase) are under way(23-25). It also possible that, to attain full cures of parasiticinfections, compounds with longer terminal half-lives than thatof E5700 will be required, and this could be a key element inlead optimization for this application.

In conclusion, our results have shown that the new quinuclidine-basedSQS inhibitors E5700 and ER-119884 have outstanding and selectiveactivities against T. cruzi in vitro. It was also found thatE5700 was capable of providing complete protection against T.cruzi infection in a murine model of fulminant acute Chagas'disease. This is the first demonstration of such antiparasiticactivity for an orally active SQS inhibitor. Thus, these quinuclidinederivatives are promising leads for the development of moreeffective and safe anti-T. cruzi agents.

ACKNOWLEDGMENTS

This work was supported by the Howard Hughes Medical Institute(grant number 55000620) and the Venezuelan Institute for ScientificResearch.

We thank Luis Alvarez for help in the preparation of figures.

FOOTNOTES

* Corresponding author. Mailing address: Instituto Venezolano de Investigaciones, Centro de Bioquimica y Biofisica, Altos de Pipe, Caracas 1020, Venezuela. Phone: (58-212)-5041660. Fax: (58-212)-5041093. E-mail: jaurbina{at}ivic.ve.

REFERENCES

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