Atovaquone Maintenance Therapy Prevents Reactivation of Toxoplasmic Encephalitis in a Murine Model of Reactivated Toxoplasmosis

T. gondii.Cysts of the ME49 strain of T. gondii were obtained from brains of NMRI mice that had been infected intraperitoneally with 10 cysts 2 to 4 months before. Mice were sacrificed by asphyxiation with CO₂, and their brains were removed and triturated in phosphate-buffered saline (PBS). An aliquot of the brain suspension was used to determine the numbers of cysts in the preparation by microscopy.

Mice and infection.Inbred female ICSBP/IRF-8^−/− mice on a C57BL/6 background were bred and maintained under specific-pathogen-free conditions in the animal facility of the Institute for Infection Medicine, Charité Campus Benjamin Franklin, Berlin, Germany. Eight- to twelve-week-old ICSBP/IRF-8^−/− mice were orally infected with 10 cysts. Mice were treated with sulfadiazine (Sigma-Aldrich, Deisenhofen, Germany) in drinking water (400 mg/liter) for 4 weeks beginning 2 days after infection to control latent infection. Two days after discontinuation of sulfadiazine, mice were treated with atovaquone nanosuspensions (10.0 mg/kg [body weight]) administered as a single i.v. dose on days 2, 5, and 8. At day 9 after discontinuation of sulfadiazine (1 day after discontinuation of acute i.v. atovaquone therapy), daily treatment with different antiparasitic drugs as maintenance therapy was initiated perorally for 1 week. At day 16 (the time point at which control mice showed symptoms of disease and/or began to succumb), the brains, livers, lungs, and sera were removed and fixed in formalin for histology or stored at −70°C for high-performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis. There were four to six mice in each experimental group to study mortality and histological changes. HPLC and MS analysis were performed on organs and serum samples of three to four mice per group. Experiments were repeated at least three times.

Atovaquone.Atovaquone (2-[trans-4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone) was obtained from Glaxo-SmithKline (Munich, Germany). Atovaquone nanosuspensions for acute i.v. therapy were produced by high-pressure homogenization under aseptic conditions. The drug powder (Glaxo-SmithKline) was dispersed in an aqueous surfactant solution containing 1% Tween 80 (ICI Surfactants, Eversberg, Belgium) by using Ultra Turrax T25 (Janke and Kunkel, Staufen, Germany). The coarse predispersion obtained was homogenized in a Micron LAB 40 high-pressure homogenizer (APV Systems, Unna, Germany) applying pressures of 150 and 500 bar (2 cycles each) and 1,500 bar (20 cycles) (11). Particles were preserved by thiomersal (Sigma-Aldrich) at a concentration of 0.001% (wt/vol). Iso-osmolarity was achieved by adjusting with glycerol (Sigma-Aldrich) at a concentration of 2.25% (wt/vol). Particle size and width of distribution (polydispersity index) were determined by photon correlation spectroscopy (Malvern Zetasizer 4; Malvern Instruments, Malvern, United Kingdom) and laser diffractometry with a Coulter LS 230 (Beckman-Coulter, Krefeld, Germany). The mean diameter measured by photon correlation spectroscopy was 459 ± 2 nm, with a polydispersity index of 0.29 ± 0.02; 99% (vol/vol) of the particles had a diameter of <2.155 μm (LD99). Atovaquone suspension (Wellvone; Glaxo-SmithKline) for oral maintenance therapy was diluted in PBS. Mice received 100, 50, or 25 mg/kg (body weight) in a total volume of 0.2 ml by gavage.

Histology.Organs were excised, fixed in a solution containing 5% formalin, and embedded in paraffin. Sagittal sections of brains and cross sections of livers and lungs were stained with hematoxylin and eosin (H&E) according to standard procedures or by an immunoperoxidase method with rabbit anti-T. gondii immunoglobulin G antibody (6). All reagents for fixing and staining with H&E were obtained from Merck (Darmstadt, Germany). For staining by the immunoperoxidase method, deparaffinized sections were incubated with swine sera at 1:10 (Dako, Carpinteria, Calif.) and with the primary antibody rabbit anti-T. gondii. For production of rabbit anti-T. gondii antibodies, rabbits were orally infected with 10 cysts of strain ME49 and treated with 300 mg of sulfadiazine/liter. Sera were harvested after a boost with T. gondii (RH strain) 15 days after infection. After a rinse with modified PBS, sections were incubated with swine anti-rabbit immunoglobulin (1:100; Dako). Finally, sections were incubated with rabbit peroxidase anti-peroxidase (1:100; Sigma-Aldrich) and with diaminobenzidine (Dako) development solution after a rinsing step. Sections stained with H&E were evaluated for inflammatory changes, and sections stained by the immunoperoxidase method were evaluated for the numbers of T. gondii cysts and T. gondii tachyzoites or antigens. The numbers of inflammatory foci and parasites were counted under ×100 magnification in three optical fields. A total of three sections of each organ from four mice in each experimental group were evaluated for the numbers of inflammatory foci and the numbers of T. gondii cysts, tachyzoites, and antigens. A modification of the score described by Araujo et al. was used (2). Briefly, normal brain was scored 1. A score of 2 reflected mild meningeal and parenchymal mononuclear cell inflammation, a score of 3 indicated severe meningeal and parenchymal inflammation, and a score of 4 was used for severe meningeal inflammation plus severe parenchymal inflammation with necrosis. The score is given as mean ± the standard deviation for at least four mice in each group.

HPLC.Weighted samples of serum (200 μl) and liver and lung (50 to 300 mg) were homogenized in 5 ml of extraction solution consisting of 2% (vol/vol) isoamyl alcohol and 98% (vol/vol) hexane in a glass-Teflon homogenizer (12). Then, 0.1 ml of serum was diluted in 5 ml of extraction solution. After the addition of 1 ml of phosphate buffer, samples were spun for 20 min in a rotating mixer (13). Suspensions were centrifuged for 10 min at 2,800 × g in a temperature-controlled centrifuge at 10°C. Then, 4 ml of supernatant was evaporated to dryness in a rotating vacuum centrifuge. The dry residue was redissolved in mobile phase (aqueous solution of 50% [vol/vol] acetonitrile and 5% [vol/vol] methanol; pH 2.65) (8, 12, 13). The samples were chromatographed on a reversed-phase column (Spherisorb C1; Waters) guarded with a C₁₈ precolumn in isocratic mode. Absorbance of the eluate was monitored at 254 nm in a UV detector (model LC 95; Perkin-Elmer, Überlingen, Germany). The linear calibration function was calculated by means of least-squares regression analysis by using computer software (SQS 98; Perkin-Elmer). The detection limit of this method was 0.6 mg/liter of serum. Limits of quantitation for tissues were ca. 0.5 mg/kg tissue. Interassay precision for serum varied from 7.4 to 15.1%. Recovery from spiked serum was 98.1 to 108.1%. Replicate extractions yielded the following extraction rates for the first extraction: 100.0% (serum), 63.6% (brain), 78.1% (liver), and 78.1% (lung).

MS.Since atovaquone concentrations were low in the brains of mice undergoing atovaquone maintenance therapy, we used MS to quantitate atovaquone in the brains of these mice. We mixed 25-μl aliquots of serum with 100 μl of internal standard solution containing mycophenolat (Roche, Grenzach, Germany) at a concentration of 0.3 mg/ml. After the addition of 300 μl of acetonitrile (VWR-International, Darmstadt, Germany), samples were vortexed and sonicated for 15 min at room temperature. Samples were then centrifuged at 13,000 × g for 6 min. The supernatants were transferred to clean tubes, and aliquots of 20 μl were injected onto a reversed-phase C₁₈ column (Eurospher; 5μ; 4.6 mm by 30 mm; Knauer, Berlin, Germany). Mobile phase A was distilled water containing 0.0025 M ammonium acetate (VWR-International). Mobile phase B was acetonitrile-ammonium hydroxide (100/0.008%). The HPLC system consisted of a Rheos 2000 HPLC pump (Flux Instruments, Basel, Switzerland) and a 233XL Autosampler (Gilson Abimed). HPLC separation was achieved with mobile-phase gradient elution (flow, 1.5 ml/min) by using the following sequence: 0 min at 100% phase A, 0.1 min at 25% phase A, 3.0 min at 25% phase A, and 3.1 min at 100% phase A. The majority (80%) of the effluent was split off before entering the MS.

An API 3000 mass spectrometer (Applied Biosystems) equipped with an electrospray ionization interface and run with Analyst 1.2 software was used for detection and quantification of atovaquone in serum and brain samples. Analytes were monitored in the negative MRM mode with the following transitions of precursor to product ions: m/z 365.1 to 171.2 (atovaquone) and m/z 318.6 to 275.2 (mycophenolat). The source temperature was set to 400°C.

Standards and quality control samples were prepared in blank mouse serum. For each batch, an eight-point standard calibration curve was analyzed; atovaquone concentrations ranged from 0.617 to 79.0 mg/ml.

For quantification of atovaquone in brain tissue the organs were weighed into polypropylene microreaction vials and homogenized mechanically using a pestle. After the addition of acetonitrile (5 μl/mg brain tissue), the samples were vigorously vortexed and sonicated for 60 min. The suspended brain tissue was sedimented at 13,000 × g for 6 min; 250-μl aliquots of the supernatant (containing the extract of 50 mg of brain tissue) were transferred to clean polypropylene vials, and 20 μl of the internal standard solution (mycophenol at 0.3 mg/ml) was added. Chromatographic and MS conditions were as described above. Six calibration standards prepared from blank mouse brain tissue were analyzed with each run. Standard concentrations ranged from 0.051 to 3.29 mg/kg.

Statistical analysis.The Fisher exact test was used to compare survival rates. Differences in numbers of inflammatory foci and parasite numbers were analyzed by using the Student t, alternate Welch t, or Wilcoxon rank-sum test.

Determination of optimal time of administration and dosage of atovaquone for acute i.v. treatment of reactivated toxoplasmosis.We have previously reported that mice acutely treated i.v. with atovaquone nanosuspensions (10 mg/kg [body weight]) do not develop reactivated toxoplasmosis, whereas all control mice died within 2 weeks after withdrawal of sulfadiazine (administered to establish latent infection) (25). To determine the maximum duration between i.v. injections of atovaquone nanosuspension, the treatment of mice with 10 mg/kg (body weight) of atovaquone every other day or every third day was compared (Table 1). Both treatment regimens showed equal therapeutic efficacies in the murine model of reactivated toxoplasmosis. While all control mice died, mice treated with 10 mg of atovaquone/kg every second or third day survived the infection (Table 1). Treated mice did not develop parasite-associated inflammatory changes in their brains (Table 1). The optimal dose of i.v. atovaquone for treatment of acute reactivated toxoplasmosis was determined by comparing treatments with 10, 5, and 2.5 mg of atovaquone nanosuspension/kg (body weight). Whereas mice treated with 10 mg of atovaquone nanosuspensions did not develop parasite-associated inflammatory changes and survived the infection, 20 and 50% of mice treated with a dose of either 5 or 2.5 mg of atovaquone nanosuspensions, respectively, developed parasite-associated inflammatory changes in their brains and died. High concentrations of atovaquone were only detectable by HPLC in the sera and organs of mice treated with 10 mg/kg (Table 1). In contrast, mice treated with 5 mg/kg showed low atovaquone concentrations in serum and liver, whereas atovaquone was not detectable by HPLC in brains. Therefore, 10-mg/kg atovaquone nanosuspensions administered every third day were used for i.v. treatment of acute reactivated toxoplasmosis in all experiments to investigate the therapeutic efficacy of a variety of antiparasitic drugs for subsequent maintenance therapy.

Murine model of reactivated TE for the evaluation of maintenance therapy.After modification of the acute treatment phase as described above, we expanded the murine model of acute reactivated toxoplasmosis to include maintenance therapy (Fig. 1). Reactivation of latent toxoplasma infection was induced by the withdrawal of sulfadiazine (used to establish latent infection in immunocompromised mice) (25). Two days later when the acute-infection therapy was reactivated in mice, atovaquone nanosuspensions were initiated every third day at a dose of 10 mg/kg (days 2, 5, and 8 after discontinuation of sulfadiazine). One day later, oral maintenance treatment with different antiparasitic drugs was started and administered daily by gavage for 7 days (Fig. 1). Sixteen days after discontinuation of sulfadiazine, sera and organs were obtained and the mortality of mice was monitored in a separate group of mice.

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FIG. 1.

Murine model of reactivated toxoplasmosis in ICSBP/IRF-8^−/− mice. After reactivation of latent toxoplasmosis, acute i.v. therapy with atovaquone nanosuspensions was given for 6 days, followed by oral maintenance therapy administered for 7 days (see Materials and Methods). d, day(s).

Effect of atovaquone maintenance therapy on survival of mice with reactivated TE.One day after completion of acute i.v. therapy with atovaquone nanosuspensions, mice were treated with different antiparasitic drugs for 7 days. Control mice began to die within 6 days after discontinuation of acute treatment; all control mice died within 9 days (Fig. 2). In contrast, all mice orally treated with atovaquone suspensions as maintenance therapy (100 mg/kg) survived the infection until the end of the observation period (10 days). The same survival rate was observed in mice treated with 50-mg/kg atovaquone suspension. The combination of pyrimethamine (0.71 mg/kg) plus sulfadiazine (30 mg/kg) administered orally (in doses equivalent to those used in AIDS patients) provided partial protection against reactivation; all mice survived the maintenance treatment period of 7 days. Starting on day 8 after initiation of maintenance treatment, these mice began to die. The mortality was 14.3% at day 10 after initiation of maintenance therapy (Fig. 2). The combination of pyrimethamine (0.71 mg/kg) plus clindamycin (35 mg/kg) showed a trend toward inferior efficacy compared to the treatment with atovaquone (P = 0.0976). Mice treated with trovafloxacin started to die at 8 days after initiation of maintenance treatment. By day 10, the mortality was 34.0%. Sulfadiazine when administered in drinking water did not protect mice against reactivation of TE (Fig. 2).

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FIG. 2.

Survival rate of ICSBP/IRF-8^−/− mice treated with atovaquone (50 or 100 mg/kg [body weight]) or control drugs. After reactivation of latent toxoplasmosis, acute i.v. therapy with atovaquone nanosuspensions was given for 6 days, followed by oral maintenance therapy administered for 7 days (see Materials and Methods). At least five mice were used in each group. The results shown are pooled from two independent experiments. Symbols: ▪, atovaquone (100 and 50 g/kg); ⊞, pyrimethamine (0.71 mg/kg) plus sulfadiazine (30 mg/kg); •, pyrimethamine (0.71 mg/kg) plus clindamycin (35 mg/kg); ○, trovafloxacin (200 mg/kg); ▿, sulfadiazine (400 mg/liter in drinking water); □, control.

Effect of atovaquone maintenance therapy on histological changes in mice with reactivated TE.Histological findings in brains and livers obtained on day 7 after initiation of maintenance therapy (the time point when maintenance therapy was stopped) paralleled the results of survival described above (Table 2 and Fig. 3). Control mice developed severe meningeal and parenchymal inflammation with numerous parasites and parasite antigens (Fig. 3A); cysts were also present in low numbers (data not shown). In the livers of control mice, we detected numerous areas of inflammation associated with parasites (Table 2). In contrast, atovaquone maintenance therapy prevented the development of TE; neither mice treated at 100 mg/kg nor those treated at 50 mg/kg showed any signs of inflammation in their brains (Fig. 3B and C) or livers. Parasites were not detectable in either organ. Similar results were obtained in mice treated with pyrimethamine plus sulfadiazine (Fig. 3D). However, signs of inflammation were observed. In contrast, the brains of mice treated with pyrimethamine plus clindamycin showed moderate meningeal and parenchymal inflammation (Fig. 3E); low numbers of parasites were detectable primarily in areas of inflammation. The numbers of inflammatory foci in the meninges and the brain parenchymas were similar in mice treated with trovafloxacin and in control mice; however, parasite numbers were significantly lower in the brains of trovafloxacin-treated mice compared to control mice (Table 2 and Fig. 3F).

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FIG. 3.

Histologic changes in brains of mice with reactivated TE at day 8 after initiation of maintenance therapy. Small arrows indicate parasitic foci (parasitophorous vacuoles and parasitic antigen); large arrows indicate inflammatory foci. Immunoperoxidase staining was performed. Magnification, ×100. (A) Control mice; (B to G) mice treated with atovaquone at 100 mg/kg (B) or at 50 mg/kg (C), pyrimethamine plus sulfadiazine (D), pyrimethamine plus clindamycin (E), trovafloxacin (F), or sulfadiazine (G). The sections shown are representative for at least four mice per group; experiments were repeated three times.

Atovaquone concentrations in serum and organs.To determine atovaquone concentrations, sera, brains, lungs, and livers were obtained on day 8 after the initiation of maintenance therapy. Atovaquone concentrations in serum samples were determined by HPLC and MS 24 h after the last administration of drug; mice orally treated with 50 or 100 mg of atovaquone/kg showed high drug concentrations of 15.00 mg/liter or higher in their sera (Fig. 4A). Atovaquone was detectable in livers and lungs at lower concentrations (Fig. 4B). The mean atovaquone concentrations in the brains of mice treated with either 50 or 100 mg/kg were 0.22 ± 0.05 or 0.34 ± 0.14 mg/kg, respectively (Fig. 4B).

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FIG. 4.

Concentrations of atovaquone in sera (A) or brains, livers, and lungs (B) of mice with reactivated TE. Mice were treated with the indicated dosages of atovaquone (50 [strippled bars] or 100 [solid bars] mg/kg) and killed 7 days after initiation of oral atovaquone maintenance therapy (1 day after the last dose of atovaquone). Atovaquone concentrations were determined by MS (sera and brains) and HPLC (livers and lungs). Values are derived from at least three mice per group and are representative for two experiments.