Host Cells Participate in the In Vitro Effects of Novel Diamidine Analogues against Tachyzoites of the Intracellular Apicomplexan Parasites Neospora caninum and Toxoplasma gondii

Abstract

The in vitro effects of 19 dicationic diamidine derivatives against the proliferative tachyzoite stages of the apicomplexan parasites Neospora caninum and Toxoplasma gondii were investigated. Four compounds (DB811, DB786, DB750, and DB766) with similar structural properties exhibited profound inhibition of tachyzoite proliferation. The lowest 50% inhibitory concentrations were found for DB786 (0.21 μM against Neospora and 0.22 μM against Toxoplasma) and DB750 (0.23 μM against Neospora and 0.16 μM against Toxoplasma), with complete proliferation inhibition at 1.7 μM for both drugs against both species. DB750 and DB786 were chosen for further studies. Electron microscopy of N. caninum-infected human foreskin fibroblast (HFF) cultures revealed distinct alterations and damage of parasite ultrastructure upon drug treatment, while host cells remained unaffected. For true parasiticidal efficacy against N. caninum, a treatment duration of 3 h at 1.7 μM was sufficient for DB750, while a longer treatment period (24 h) was necessary for DB786. Pretreatment of tachyzoites for 1 h prior to host cell exposure had no effect on infectivity. However, pretreatment of uninfected host cells had a significant adverse effect on N. caninum proliferation: exposure of HFFs to 1.7 μM DB750 for 6, 12, or 24 h, followed by infection with N. caninum tachyzoites and subsequent culture in the absence of DB750, resulted in significantly delayed parasite proliferation. This suggests that either (i) these compounds or their respective active metabolites were still present after the removal of the drugs or (ii) the drug treatments reversibly impaired some functional activities in HFFs that were essential for parasite proliferation and/or survival.

Neospora caninum is one of the most important causative agents of infectious bovine abortion, stillbirth, and the birth of weak calves; as such, it represents a serious economic and veterinary medical problem (18). In addition, N. caninum causes neuromuscular disease in dogs, and neosporosis has been demonstrated in a wide range of other species worldwide (17, 29). Neospora caninum is closely related to Toxoplasma gondii, the causative agent of toxoplasmosis in humans and many other animal species (31). Neospora and Toxoplasma share many morphological and ultrastructural features. They are both able to invade many different cell types in vitro, and they synthesize many highly homologous, and probably functionally related, proteins. Nevertheless, the two species clearly differ in other features of their biology, pathology, and host-parasite interaction (reviewed in references 32 and 35).

The economic impact of neosporosis on the cattle industry is rather extensive (26, 27; for a review, see reference 18). It has been estimated that in California approximately 40,000 abortions could be due to neosporosis, providing an estimated loss of 35 million U.S. dollars per year (5). Besides the loss caused by the abortion itself, reduced milk yield (33, 34), premature culling (53), and reduced postweaning weight gain in beef calves (4) have to be considered. In Australia and New Zealand, losses are thought to be more than 100 million Australian dollars per year (48, 49), and in The Netherlands, estimates of 19 million euros have been reported. A recent study estimating the median annual losses due to N. caninum in the Swiss dairy cow population led to a result of 9.7 million euros (26). Thus, the economic importance of neosporosis, especially in cattle, has led to research on the development of strategies for the prevention and treatment of N. caninum infection. Vaccination has been proposed as a potential option for the prevention of abortion, and a number of promising vaccine candidates and vaccination approaches have been established; however, no valuable vaccine capable of preventing endogenous transplacental infection is available to date (reviewed in references 18, 32, and 36).

More recently, chemotherapy has been identified as an economically promising option (26, 27), provided an effective drug can be found. A wide range of compounds, including lasalocid, monensin, piritrexim, pyrimethamine, clindamycin, robenidine, and trimethoprim, have been shown previously to exhibit proliferation-inhibitory activity against N. caninum tachyzoites in cell culture-based assays (40, 42). More recently, artemisinin (37), depudecin (39), toltrazuril, ponazuril (14), nitro-and bromothiazolides (20, 21, 22), and alcoholic herbal extracts (55) have also been reported to exhibit antiparasitic activity against tachyzoites in cell culture. Only a few drugs have been evaluated in small-animal models. Sulfadiazine and amprolium were investigated, and sulfadiazine administered at 1 mg/ml prevented disease in experimentally infected mice but did not eliminate the parasite (41). Several studies with mice focused on toltrazuril (1, 23, 24), showing that inclusion of toltrazuril in the drinking water eliminated parasites in the central nervous system but that cell-mediated immunity was required in order for toltrazuril to achieve full efficacy in mice. In addition, toltrazuril treatment controlled diaplacental N. caninum transmission in experimentally infected pregnant mice. Studies on prophylactic toltrazuril administration to newborn calves suggested that this treatment regime could exhibit a certain degree of protective efficacy (25, 38). Treatments of dogs with clindamycin, potentiated sulfonamides, and pyrimethamine, as reported by Barber and Trees, were successful in eliminating clinical signs in 10 of 27 cases of canine neosporosis (3).

Diamidines represent a class of broad-spectrum antimicrobial compounds, of which pentamidine and its analogues exhibit activity against intracellular and extracellular protozoan parasites (reviewed in reference 10). Since its discovery, pentamidine has been the most widely used diamidine and has been successfully applied to treat a variety of parasitic infections, including African trypanosomiasis, leishmaniasis, and malaria. With the emergence of AIDS and the frequent association of AIDS with Pneumocystis carinii infections, this class of drugs has become even more popular (reviewed in reference 54). Other diamidine-containing drugs, such as diminazene aceturate, are commonly used for trypanosome chemotherapy of livestock, but this drug is prone to resistance formation. The development of novel pentamidine analogues led to derivatives that exhibited a more favorable pharmacokinetic profile, improved bioavailability, lower toxicity, and a higher chance of passing the blood-brain barrier (11). Currently, one of the prodrugs of furamidine (DB289) is in phase III clinical trials for human African trypanosomiasis, malaria, and Pneumocystis carinii pneumonia. Other analogues, such as dicationic furans and dicationic carbazole compounds, have been reported to exhibit good efficacy against Cryptosporidium parvum in neonatal ICR mouse models (8, 9). In this study, we describe the effects of new diamidines against N. caninum and T. gondii tachyzoites grown in fibroblast cell culture, and we demonstrate that the host cell participates in the in vitro effects of these drugs.

MATERIALS AND METHODS

Culture media, buffers, and reagents.

Unless otherwise stated, all tissue culture media were purchased from Gibco-BRL (Zurich, Switzerland), and biochemical reagents were from Sigma (St. Louis, MO). The diamidine derivatives used in this study were synthesized at the Department of Chemistry and the Center for Biotechnology and Drug Design, Georgia State University. They were kept as dry powders or as stock solutions of 1 mg/ml in dimethyl sulfoxide (DMSO) and were stored at −20°C.

Cell culture and parasite purification.

Vero cells were maintained in RPMI 1640 medium supplemented with 5% fetal calf serum (FCS), 2 mM l-glutamine, 50 U of penicillin/ml, and 50 μg of streptomycin/ml at 37°C under 5% CO₂ in tissue culture flasks and were trypsinized three times a week. Human foreskin fibroblasts (HFF) were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% FCS, 50 U of penicillin/ml, and 50 μg of streptomycin/ml at 37°C under 5% CO₂ in tissue culture flasks. Cultures were trypsinized once a week. N. caninum tachyzoites of the Liverpool isolate (2) and T. gondii RH tachyzoites were cultured in Vero cell monolayers, with FCS being replaced by 5% immunoglobulin-free horse serum (30). Intracellular parasites were harvested by trypsinization of infected Vero cells, followed by repeated passages through a 25-gauge needle at 4°C and separation from cell debris on Sephadex-G25 columns as described previously (30). Purified tachyzoites were used to infect HFF monolayers as described below.

In vitro drug treatment assays for N. caninum and T. gondii.

In vitro drug treatment assays were carried out in triplicate essentially as described previously (20, 21, 22). HFF were grown to confluence either in the presence or in the absence of glass coverslips in 24-well tissue culture plates, and each well was infected with 5 × 10⁴ cell culture-derived, freshly purified N. caninum or T. gondii tachyzoites resuspended in DMEM containing 5% FCS, 50 U of penicillin/ml, and 50 μg of streptomycin/ml. Following incubation for 1 h at 37°C under a 5% CO₂ atmosphere, unbound parasites were removed by washing in DMEM, and 1 ml of DMEM-FCS-penicillin-streptomycin was added, containing the compounds at the concentrations indicated in the individual experiments. For a list of some of these compounds, see Table 1. In some experiments, compounds were added only after 2 or 3 days of culture (to assess the effects on established cultures). Each experiment included controls such as (i) parasite-infected HFF in media containing concentrations of the DMSO solvent corresponding to drug concentrations and (ii) uninfected HFF monolayers in drug-containing medium to assess selective toxicities. The cultures were maintained at 37°C under a 5% CO₂ atmosphere for different periods as indicated below and were inspected daily by light microscopy.

TABLE 1.

Structures of reversed amidines and efficacies^a against Neospora caninum and Toxoplasma gondii tachyzoites in vitro^b

^aExpressed as IC₅₀s.

^bHFF monolayers were infected with tachyzoites, and samples were taken after 3 days and analyzed by real-time PCR. IC₅₀s were calculated after the logit-log-transformation of relative growth (see Materials and Methods).

^cMW, molecular weight.

^dND, not determined.

Primary evaluation of drug efficacy at concentrations of 5, 1, and 0.2 μg/ml after 3 days of culture was done light microscopically following staining with cresyl violet. Efficacies were assessed by counting the numbers of parasites in 10 randomly chosen fields per sample. Samples were scored as either − (no visible tachyzoites), + (1 to 50 tachyzoites), ++ (50 to 500 tachyzoites), or +++ (more than 500 tachyzoites).

Fifty percent inhibitory concentrations (IC₅₀s) of selected compounds (DB811, DB786, DB766, and DB750) were determined in cultures treated with drugs at different concentrations, ranging from 0.085 to 3.4 μM, and in control cultures without drugs, after 3 days following infection. Samples for quantitative Neospora and Toxoplasma real-time PCR analysis were taken by removal of the medium and addition of a mixture of 200 μl of phosphate-buffered saline (PBS), 180 μl of lysis buffer, and 20 μl of proteinase K (DNeasy kit; Qiagen, Basel, Switzerland). Samples were frozen at −20°C until further processing. IC₅₀s were calculated after the logit-log transformation of relative growth (RG; considered to be 1 for the control) according to the formula ln[RG/(1 − RG)] = a × ln(drug concentration) + b (where a is the increase of the linear graph and b is the interception point corresponding to the IC₅₀) and subsequent regression analysis by the corresponding software tool contained in the Excel software package (Microsoft, Seattle, WA).

To determine the time span required for DB786 and DB750 to exert a true parasiticidal effect, freshly infected HFF cultures were treated with 1.7 μM each drug for 3 h, 6 h, 12 h, 24 h, or 48 h. The drug-containing medium was then removed and replaced with fresh culture medium without drug. To monitor subsequent tachyzoite proliferation, samples were taken at different time points up to 35 days (DB786) or 21 days (DB750) posttreatment and were analyzed by real-time PCR. During this time span, the culture medium was changed every 7 days, and the cells were trypsinized and reseeded once, at day 18.

Treatments of N. caninum tachyzoites prior to infection.

N. caninum tachyzoites (5 × 10⁴) were resuspended in 100 μl of DMEM containing 5% horse serum and were incubated with 1.7 μM DB786 or DB750 for 2 h or 6 h at 37°C. The suspension was then added to the HFF monolayers (in 1 ml final culture medium) for 30 min at 37°C under 5% CO₂. The pyrrolidine dithiocarbamate-based adhesion/invasion assay (47) was used to monitor the effects of drugs on the invasive capacities of drug-treated versus untreated tachyzoites. For this purpose, unbound parasites were removed by a wash in DMEM, and infected monolayers were incubated with DMEM containing 100 μM pyrrolidine dithiocarbamate, 0.2 μM CuSO₄, and a polyclonal rabbit hyperimmune serum raised against entire N. caninum tachyzoites (1:200) for 2 h at 37°C. In parallel, control incubations in DMEM were performed. Subsequently, the wells were washed once with DMEM, and DMEM containing 1 mg/ml DNase I was added. The preparations were incubated for 1 h at 37°C. Control wells were also washed and incubated with DMEM. Finally, all wells were washed with medium containing 1 mM EDTA to inhibit DNase I activity, and the cellular material was taken up in 180 μl of lysis buffer (DNeasy kit; Qiagen). The specimens were transferred to Eppendorf tubes, heated for 5 min at 95°C, and stored at −20°C prior to further use.

Treatments of HFF host cells.

In some experiments, drug treatments were initiated prior to infection with tachyzoites. For this purpose, confluent HFF were incubated in medium containing 1.7 μM DB750 or DB786 for 6 h, 12 h, or 24 h. Subsequently the medium was removed, the cells were washed three times for 20 min each time in DMEM at 37°C, and the monolayers were infected as described above. In some experiments, the treated monolayers were maintained in culture medium for 24 h, 48 h, or 72 h prior to infection. Controls were performed without drug pretreatment, but corresponding concentrations of DMSO were added. Samples were collected for real-time PCR quantification of parasite proliferation at different time points as indicated below.

To monitor the direct effects of DB750 and DB786 on the adhesive capacity and growth of HFF, confluent HFF lawns were trypsinized, and cells were resuspended in fresh medium and transferred to 24-well-plates (5 × 10³ cells per well) containing the drugs (at 1.7 μM) or DMSO as a solvent control. After 2 and 12 h, the adherent HFF in 20 different fields were counted. At days 2 and 3, the medium was removed, and attached cells were washed with PBS, trypsinized, and counted using a Neubauer chamber. For determination of the effects of DB768 and DB750 on confluent HFF monolayers, cells were transferred to 24-well-plates and grown to 100% confluence for 6 days. Then the medium was replaced with fresh medium containing 1.7 μM DB786 or DB750 or corresponding amounts of DMSO. After 72 h, the medium was removed, and attached cells were washed with PBS, trypsinized, and counted using a Neubauer chamber.

Processing of DNA samples and LightCycler-based quantitative PCR.

DNA was purified with the DNeasy kit (Qiagen) according to the protocol for tissue samples. Samples were then eluted in a volume of 100 μl of AE buffer and boiled for 5 min. For N. caninum, detection of DNA amplification products and quantification of parasite numbers through fluorescence resonance energy transfer were performed on the LightCycler instrument (Roche Diagnostics, Basel, Switzerland) as previously described (46). LightCycler-based quantification of T. gondii proliferation was done according to the work of Costa et al. (13) and Scheidegger et al. (50).

As external standards, samples containing the DNA from 10, 100, and 1,000 N. caninum or T. gondii tachyzoites were included. The parasite count for a given sample was calculated by interpolation from this standard curve. Each assay in a given experiment was carried out in quadruplicate, and the outcome of one representative experiment of at least three independent experiments, all producing virtually identical results, is shown.

TEM.

HFF cell layers were grown to confluence and infected with N. caninum tachyzoites, and at days 2 and 3 postinfection, treatments with DB750 and DB786 (1.7 μM) were initiated. After 4, 24, and 72 h, samples were collected by removal of the medium, briefly washed in 100 mM sodium cacodylate buffer (pH 7.2), and fixed in cacodylate buffer containing 2.5% glutaraldehyde. Cells were scraped off with a rubber policeman and centrifuged for 10 min at 4°C and 1,000 × g, and the resulting pellet was further fixed at 4°C overnight, followed by postfixation in 1% OsO₄ in cacodylate buffer for 4 h at 4°C. Subsequently, specimens were washed in water and prestained in 1% uranyl acetate in water for 1 h at 4°C, followed by extensive washing in water. Following dehydration in a graded series of ethanol (30, 50, 70, 90, and 100%), they were embedded in Epon 820 epoxy resin over a period of 2 days with three resin changes. The resin was polymerized at 65°C for 24 h. Ultrathin sections were cut on a Reichert and Jung ultramicrotome and were loaded onto 300-mesh copper grids (Plano GmbH, Marburg, Germany). Staining with uranyl acetate and lead citrate was performed as described elsewhere (28, 31). Grids were viewed on a Philips 400 transmission electron microscope (TEM) operating at 80 kV.

Statistical analysis.

The significance of the differences between the end point values of the control and experimental assays in the growth and inhibition experiments was determined by Student's t test using the Microsoft Excel program. P values of <0.05 were considered statistically significant.

RESULTS

A defined subset of diamidines acts against the intracellular apicomplexan parasites N. caninum and T. gondii.

Nineteen different diamidine compounds, including six reversed amidines, were tested for their activities against N. caninum tachyzoites in cell culture. Compounds were added at different concentrations either every day, with fresh compound in medium, or only once, at the beginning of treatment. The two procedures yielded identical results (data not shown). Primary light microscopic assessment showed that four of the six reversed amidines, namely, DB811, DB786, DB766, and DB750 (Table 1), exhibited clear-cut proliferation-inhibitory potential at the lowest concentration. None of the other diamidines were effective, even at concentrations as high as 5 μg/ml. The four effective reversed amidines all represent modified versions of DB75 (furamidine) (Table 1) and are built symmetrically, with the two core structure-benzene rings being altered through the addition of either two chloro atoms (DB811), two hydroxy groups (DB750), or iso-propoxy groups at different positions (Table 1).

The IC₅₀s of DB786, DB750, and DB766 were found to be in the same range (0.21 μM, 0.23 μM, and 0.30 μM, respectively), while that of DB811 was slightly higher (0.66 μM) (Table 1). Similar results were obtained when these compounds were assessed for activity against T. gondii: the IC₅₀s of DB750 and DB786 were 0.16 μM and 0.22 μM, respectively, demonstrating that these compounds were active against both Neospora and Toxoplasma (Table 1). None of the four compounds exhibited any adverse effects on the morphology of HFF monolayers during these experiments.

DB786 and DB750 were the most efficient antiparasitic compounds and had no notable effects on HFF host cell viability and growth when applied at a concentration (1.7 μM) that completely blocked tachyzoite proliferation. Following trypsinization, HFF adhered readily to the plastic surfaces of tissue culture devices in the presence of both drugs and proliferated with a speed similar to that of untreated HFF; moreover, there was no impairment of confluent monolayers even in the presence of DB786 or DB750 for extended periods (up to 6 days) (data not shown). Thus, both DB786 and DB750 exhibited selective toxicity against N. caninum and T. gondii at submicromolar levels. Subsequent studies focused on these two drugs, and mainly on their activity against N. caninum.

Characterization of the antiparasitic activities of DB786 and DB750.

We determined the minimal amount of time required for the drugs to act permanently on the parasites in order to exert a true parasiticidal effect. N. caninum-infected monolayers were treated with 1.7 μM DB750 and DB786 for various time spans ranging from 3 to 72 h, followed by further culture without the drugs for 3 weeks (Fig. 1). We found that under these conditions, the Neospora-infected HFF needed to be in contact with DB750 for only 3 h, and this contact eliminated any further parasite proliferation during the 3-week follow-up period (Fig. 1A). In contrast, DB786 required a treatment duration of 24 h for the proliferation-inhibitory effect to be maintained (Fig. 1 B). Thus, the selective antiparasitic toxicities of these two compounds act rather rapidly and are highly efficient.

FIG. 1.

Parasiticidal efficacies of DB750 (A) and DB786 (B) against N. caninum tachyzoites. (A) Growth curves after treatments of infected HFF with DB750 (1.7 μM) for 3 to 24 h. Note that an incubation period of 3 h is sufficient to exert a highly significant parasiticidal effect (P < 0.05). (B) Growth curves after treatments of infected HFF with DB786 (1.7 μM) for 3 to 48 h. Note that a 24-h incubation period is required to definitely stop parasite proliferation. Data are means from experiments performed in triplicate plus standard deviations. Results representative of three independent experiments are shown. IC, infection control; p.i., postinfection.

We then investigated whether DB750 and DB786 would also be effective if drug treatment was initiated at later stages of infection, when larger pseudocysts containing numerous tachyzoites had already formed. Figure 2 demonstrates that each of the two drugs, applied at 1.7 μM, exhibited a massive and rapid inhibitory effect at days 2 and 3 of infection.

FIG. 2.

In vitro treatments of established N. caninum infections. Tachyzoites were allowed to proliferate in HFF for 2 or 3 days (arrows), and then DB750 or DB786 treatment (each at 1.7 μM) was initiated. Note the immediate halt in tachyzoite proliferation after the addition of the drugs, followed by a decrease in parasite numbers with time. Data are means from experiments performed in triplicate plus standard deviations. Results representative of three independent experiments are shown. Parasite numbers in treated samples at day 10 are significantly lower than tachyzoite numbers in the infection control (IC) at day 4 (P < 0.05). p.i., postinfection.

The morphological and structural alterations associated with DB750 and DB786 treatments of N. caninum-infected HFF were identified by TEM. In untreated control cultures (Fig. 3), N. caninum tachyzoites formed well-defined parasitophorous vacuoles surrounded by a parasitophorous vacuole membrane and containing variable numbers of tachyzoites, embedded in a tubular membranous network that provides the vacuole matrix. No differences from control cultures were noted in drug-treated samples after 3 h (data not shown). However, upon exposure to DB750 (Fig. 4) and DB786 (data not shown) for 24 to 48 h, dramatic alterations were noted. These concerned the occurrence of increasing numbers of vacuoles within the parasite cytoplasm (Fig. 4A to C). These vacuoles appeared empty or were filled with membranous and electron-dense material of unknown origin. In some cases, nuclear chromatin condensation was noted (Fig. 4C), but the parasite plasma membrane still remained intact. The parasitophorous vacuole matrix containing the tubular membranous network either was lacking in drug-treated cultures (Fig. 4D) or was replaced by irregular membranous residues (Fig. 4B). The dying parasites were still separated from the host cell cytoplasm by the parasitophorous vacuole membrane, but often additional layers of membrane, surrounding the entire vacuole, were noted (Fig. 4C). At 48 h (Fig. 4D to F), alterations became more prominent: vacuoles and lipid droplets formed within and around the tachyzoites (Fig. 4D), the cytoplasm of parasites was filled with electron-dense material of a rather amorphous appearance (Fig. 4E), and an increasing number of extracellularly located tachyzoite ghosts became visible (Fig. 4F). Similar damage was induced upon treatment of T. gondii tachyzoites with DB750 (Fig. 5) or DB786 (data not shown). In accordance with the observations that the drugs affected neither proliferating nor confluent HFF cells, no alterations in the fine structure were observed in HFF with either of the two drugs (data not shown).

FIG. 3.

TEM of N. caninum-infected HFF cultured in the absence of drugs. Note the presence of tachyzoites within a parasitophorous vacuole, surrounded by a defined parasitophorous vacuole membrane. (A) Single tachyzoite. mito, mitochondrion; nuc, nucleus. Bar, 0.6 μm. (B) Larger vacuole containing several tachyzoites embedded within the parasitophorous vacuole tubular network (pvtn). Bar, 0.75 μm. (C) Larger vacuole with numerous tachyzoites. Bar, 1.25 μm.

FIG. 4.

TEM of DB750-treated N. caninum tachyzoite-infected HFF after 24 h (A to C) and 48 h (D to F) of drug exposure. Hallmarks in changes are increased vacuolization of the parasite cytoplasm (vac), alterations within the vacuolar matrix and loss of parasitophorous vacuole tubular network (pvtn) organization (B), and in some cases, increased membrane accumulation surrounding the parasitophorous vacuole (arrows) (C). nuc, nucleus. Other features are the accumulation of lipid droplets surrounding the dying parasites (asterisks) (D) and electron-dense deposits within the tachyzoite cytoplasm (E). Often, extracellular and heavily damaged tachyzoites were observed (F). Bars, 0.45 μm (A), 0.6 μm (B), and 0.5 μm (C to F).

FIG. 5.

TEM of T. gondii-infected HFF (A) and DB750-induced damage (B and C). Note the vacuolization (vac) and loss of structural integrity in drug-treated parasites. nuc, nucleus; pvtn, parasitophorous vacuole tubular network. Bars, 0.3 μm.

Further experiments were performed in order to investigate whether DB786 and DB750 could affect host cell entry by N. caninum tachyzoites. For this purpose, freshly purified tachyzoites were incubated in medium containing the drugs for 2 h or 6 h prior to host cell infection. Tachyzoites maintained for 6 h in the absence of any drugs exhibited an approximately 50% reduction in infectivity due to the fact that extracellular maintenance of these parasites has harmful effects, confirming earlier observations (20, 22, 47). However, quantitative real-time PCR showed that treatments with DB786 and DB750 had no impact on the host cell interaction, suggesting that the drugs affected only intracellular, not extracellular, tachyzoites (data not shown).

Pretreatment of uninfected HFF prior to infection has a “memory effect” and severely impairs parasite growth.

Uninfected HFF monolayers were treated with DB750 or DB786 for 24 h, washed in medium without drugs three times for 20 min each time at 37°C, and then infected with N. caninum tachyzoites and cultured in the absence of the drugs. Surprisingly, no proliferation of tachyzoites was noted for 7 days (data not shown). In order to investigate how long this “memory effect” would last, the experiment was repeated with a modification, including extended periods (24, 48, and 72 h) for washing between treatment and infection (Fig. 6A). The proliferation-inhibitory effect was still evident after a 2-day period of washing in medium (Fig. 6A), but after 3 days, the proliferation-inhibitory effect was lost. Identical results were achieved with DB750 (Fig. 6A) and DB786.

FIG. 6.

“Memory effect” after treatment of HFF with DB750 (1.7 μM) prior to infection with N. caninum tachyzoites severely impairs parasite proliferation. (A) After a 24-h treatment with DB750, inhibition of tachyzoite proliferation is maintained for 48 h. After 72 h, proliferation starts to occur. IC, infection control; p.i., postinfection. (B) Inhibition of tachyzoite proliferation is maintained for 7 days irrespective of whether the treatment period is short (6 h) or long (12 to 24 h). Data are means from experiments performed in triplicate plus standard deviations. Results representative of three independent experiments are shown.

In order to determine the shortest necessary period of host cell pretreatment that would confer extended protection from parasite proliferation, HFF monolayers were treated with DB750 (1.7 μM) for 6 h, 12 h, or 24 h; washed three times, for 20 min each time, in medium; infected with N. caninum tachyzoites; and further cultured in the absence of the drug. We found that irrespective of the duration of treatment, the proliferation-inhibitory effect lasted 7 days, and only at later time points was a slow and modest increase in parasite numbers noted (Fig. 6B). This increase in tachyzoite numbers was more pronounced in samples that were treated for shorter periods of time.

DISCUSSION

Four out of 19 diamidines (DB750, DB766, DB786, and DB811) exhibited profound in vitro activities against N. caninum and T. gondii tachyzoites. In vitro efficacies of pentamidine and nine pentamidine analogues against T. gondii have been reported previously (42), and some pentamidine analogues exhibited proliferation-inhibitory properties at concentrations around 10 μg/ml. Hexamidine and 1,4-di[4-(2-imidazolinyl)-2-methoxy-phenoxy]butane were active in a similar concentration range (42). In contrast, DB750, DB766, DB786, and DB811 exhibited submicromolar IC₅₀s ranging from 0.160 μM to 0.660 μM (0.14 to 0.5 μg/ml). These four compounds are structurally related in that they are derived from furamidine, a bis-amidine diphenylfuran derivative, with hydrophobic moieties substituting the amidine ends of the molecules (Table 1). However, at concentrations up to 17 μM, furamidine was ineffective against N. caninum and T. gondii (data not shown).

It was reported previously that N-phenyl substitution of furamidine markedly increased its antiparasitic activity against the intracellular stages of Trypanosoma cruzi and Leishmania amazonensis, with IC₅₀s in the lower micromolar (2 to 4 μM) range (15). In addition, phenyl-substituted furamidine caused apoptosis-like death of T. cruzi (16). Thus, it is likely that the addition of hydrophobic groups at the two ends of the molecule increases membrane permeability. Prospectively, this could also increase the bioavailability of these compounds in vivo. This is important for apicomplexan parasites such as N. caninum and T. gondii, because any compound that needs to target these organisms has to cross at least three distinct membrane layers: the host cell membrane, the membrane of the parasitophorous vacuole, and the parasite plasmalemma. In addition, depending on the mechanism(s) of action, other organellar membranes (mitochondria, Golgi complex) could also get involved. However, at present we do not know how the drugs traverse these compartments, and how they reach these intracellular parasites remains to be investigated.

In other parasitic infections, diamidine uptake has been found to be based mainly on specific accumulation of these drugs by the parasite due to high-affinity transporters. For instance, for African trypanosomes, the P2 nucleoside transporter (12) was found to cause a 1,000-fold-higher accumulation of the drug within the parasite than in the medium. Later, two further high- and low-affinity transporters, HAPT1 and LAPT1, respectively, were discovered (10). In Leishmania mexicana, intracellular drug levels similar to those in African trypanosmes are not reached (6). A divalent cation channel may be involved in the transport of pentamidine into these parasites (10). Comparison of pentamidine-resistant and wild-type leishmaniae indicated that the drug accumulates, and exerts its action, in the mitochondria of wild-type, but not of resistant, parasites. Other studies have suggested that mitochondrial uptake is driven by the higher mitochondrial membrane potential (MMP). Treatment of leishmaniae with pentamidine leads to a collapse in the MMP and disintegration of the kinetoplast network (6, 10). In Plasmodium falciparum-infected erythrocytes, pentamidine levels are up to 500 times higher than those in the surrounding medium or uninfected erythrocytes, and diamidines might enter the parasite by a choline transporter. Stead et al. (52) suggested that pentamidine binds to ferriprotoporphyrin IX (FPIX), which is generated during hemoglobin digestion. The binding to FPIX then probably kills the parasite by inhibiting the crystallization of the otherwise toxic FPIX.

Diamidines have been shown to exhibit DNA-binding properties. In African trypanosomes, diamidines disrupt the kinetoplast within the single mitochondrion and cause enlargement of the mitochondrion and disruption of the MMP (43). Other mechanisms of action include disturbance of polyamine metabolism (7), inhibition of peptidase activity (45), and interference with normal topoisomerase II activity (51). Some diamidines, such as furamidine and its analogues, exhibit fluorescent properties and have been directly used for localizing the major site of action within the cell. In T. cruzi-infected cardiomyocytes and macrophages, DB75 and its phenyl-substituted analogue DB569 were found to be associated with the nucleus and the kinetoplast (15). Neither DB750 nor DB786 exhibits fluorescent properties; thus, it was not possible, by fluorescence microscopy, to investigate which cellular compartments within N. caninum and T. gondii tachyzoites were affected. Since these molecules have been specifically designed to bind to AT-rich DNA (19, 44), it is conceivable that they also do so in these apicomplexans. However, some researchers suggest that the DNA-binding properties of diamidines are a critical, but probably not an exclusive, requirement for antiparasitic activity (54).

Ultrastructural studies of T. cruzi bloodstream forms treated with furamidine and a phenyl-substituted furamidine analogue had earlier revealed distinct alterations such as enlargement of the mitochondrion, fragmentation of the kineoplast, and changes in the nucleus as early as 2 h after the initiation of drug treatment (15, 16). In contrast to T. cruzi trypomastigotes, extracellular N. caninum tachyzoites were not affected by DB750 or DB786, since neither adhesion nor invasion of HFF was reduced upon treatment of extracellular parasites for as long as 6 h. This finding indicates that these drugs are not effectively taken up when parasites do not reside within a host cell, or it could imply that the biochemical pathways affected by these drugs are most likely not active in extracellular parasites. TEM of intracellular N. caninum and T. gondii revealed no obvious alterations in parasite ultrastructure as early as after 3 h of drug treatment. However, clear alterations, such as increased cytoplasmic vacuolization and membrane alterations within the lumen of the parasitophorous vacuole, were evident at 24 h, and the occurrence of electron-dense bodies and crystalline structures at 48 h demonstrated the damage induced upon drug treatment.

A number of observations point toward an involvement of the host cell in the process of parasite killing by DB750 and DB786. First, by TEM, it was evident that the parasitophorous vacuole was in many instances surrounded by several layers of membrane, and often lipid droplets were seen adjacent to the parasitophorous vacuole membrane (Fig. 4 and 5). Second, the duration of treatment necessary for DB750 to exert a parasiticidal effect was 3 h (Fig. 1), despite the fact that no obvious ultrastructural damage was visible at that time. Third, DB750 pretreatment (1.7 μM) of HFF monolayers for 24 h prior to infection with N. caninum also resulted in severe growth inhibition for as long as 1 week. This indicates that the host cells most likely incorporated these compounds, or active metabolic products, for extended periods, creating a “memory effect” that could last for as long as a week, as evidenced in this study. Nevertheless, HFF remained unaffected by these compounds. Whether HFF cells do indeed incorporate these drugs, how the host cells acquire these compounds (whether through active uptake by specific transporters or through simple diffusion), and where these compounds are stored will be investigated in the future. To our knowledge, no such effect has been described for related drugs currently in use for other parasites. However, it is noteworthy that pretreatment of host cells was not parasiticidal, allowing tachyzoites to resume proliferation after some time.

In conclusion, novel diamidine compounds, most notably DB786 and DB750, exhibit in vitro activities against N. caninum and T. gondii in the submicromolar range. The activities of these drugs are limited to intracellular parasites, and it appears that the host cell metabolism is involved in keeping parasite proliferation under control. These findings render DB750 and DB786 promising candidates for further studies in vivo.