Standort in Deutschland, wo man günstige und qualitativ hochwertige Kamagra Ohne Rezept Lieferung in jedem Teil der Welt zu kaufen.
Kaufen priligy im Online-Shop.
Wirkung ist gut, kommt sehr schnell, innerhalb von 5-7 Minuten. propecia was nur nicht versucht, verbrachte eine Menge Geld und Nerven, und geholfen hat mir nur dieses Tool.
Atovaquone maintenance therapy prevents reactivation of
toxoplasmic encephalitis in a murine model of reactivated
ILDIKO R. DUNAY1,2, MARKUS M. HEIMESAAT1, FARIS NADIEM BUSHRAB3, RAINER
H. MÜLLER3, HARTMUT STOCKER4, KEIKAWUS ARASTEH4, MICHAEL KUROWSKI5,
RUDOLF FITZNER6, KLAUS BORNER6, and OLIVER LIESENFELD1,*
Institute for Infection Medicine, Department of Medical Microbiology and Immunology of
Infection, Charité Campus Benjamin Franklin, Hindenburgdamm 27, D-12203 Berlin, Germany1,
Semmelweis University Budapest, Nagyvàrad tèr 4, 1089 Budapest, Hungary2, Department of
Pharmaceutics, Biotechnology and Quality Management, Free University of Berlin, Kelchstr. 31,
D-12169 Berlin, Germany3, Vivantes Auguste-Viktoria Klinikum, Rubensstrasse 125, 12157
Berlin, Germany4, HIV-Lab, c/o Vivantes Auguste-Viktoria Klinikum, Rubensstrasse 125, 12157
Berlin, Germany5, Institute of Clinical Chemistry and Pathobiochemistry, Charité Campus
Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany6
* To whom correspondence should be addressed
Atovaquone maintenance therapy for reactivated toxoplasmosis
Acute therapy with pyrimethamine plus sulfadiazine is the treatment of choice for reactivated
toxoplasmic encephalitis (TE). Acute therapy is followed by lifelong maintenance therapy
(secondary prophylaxis) with the same drugs at lower dosages. The use of pyrimethamine plus
sulfadiazine is hampered by severe side effects including allergic reactions and hematotoxicity.
Alternative treatment regimens with pyrimethamine plus clindamycin or other antiparasitic drugs
are less efficacious. Atovaquone nanosuspensions show excellent therapeutic effects for “acute”
i.v. treatment of reactivated TE in a murine model. In the present study, the therapeutic efficacy
of atovaquone for oral “maintenance” therapy was investigated. Mice with a targeted mutation in
the interferon regulatory factor 8 gene were latently infected with Toxoplasma gondii, developed
reactivated TE, and received acute i.v. therapy with atovaquone nanosuspensions. Mice were then
treated orally with atovaquone suspension or other antiparasitic drugs to prevent relapse of TE.
Maintenance therapy with atovaquone at daily doses of 50 or 100 mg/kg body weight protected
mice against reactivated TE and death. This maintenance treatment was superior to standard
therapy with pyrimethamine plus sulfadiazine. The latter combination was superior to the
combination of pyrimethamine plus clindamycin. Inflammatory changes in the brain parenchyma
and menings as well as parasite numbers in brains of mice confirmed the therapeutic efficacy of
atovaquone for maintenance therapy. Atovaquone was detectable in serum, brains, livers, and
lungs of infected mice by HPLC and/or mass spectrometry. In conclusion, atovaquone appears to
be superior to the standard maintenance therapy regimens in a murine model of reactivated TE.
The therapeutic efficacy of atovaquone for maintenance therapy against TE should be further
(T.g.) is an intracellular protozoan parasite of human and animals with
worldwide distribution. Seroprevalence varies with geographical location and up to 70% in
Germany and France [1, 2]. Following initial uptake of the parasite in the gut and dissemination
throughout the body, the latent stage of infection is characterized by the presence of parasites in
cysts in the central nervous system and muscle tissues . Immunocompromised hosts, i.e.
patients with AIDS or organ transplant recipients, are at risk of reactivation of the infection by
rupture of cysts . Toxoplasmic encephalitis (TE) is the most common clinical manifestation of
reactivated disease in AIDS patients who do not receive highly active antiretroviral therapy
HAART or antiparasitic prophylaxis TE is the most frequent infectious cause of focal
intracerebral lesions in these patients [2-4]. Untreated, reactivation of disease leads to death of
the patient. The acute therapy (pyrimethamine plus sulfadiazine) of TE is followed by lifelong
maintenance therapy [2, 5]. The standard regimen for maintenance therapy includes
pyrimethamine plus sulfadiazine at lower dosages . Pyrimethamine plus sulfadiazine therapy is
hampered by severe side effects including hematologic toxicity and/ or life-threatening allergic
reactions in between 5 and 15% of patients [5, 6].
The hydroxynaphthoquinone atovaquone is a potent inhibitor of the respiratory chain of parasites
with potent in vitro and in vivo activity against both the tachyzoite and cyst forms of T. gondii
11]. The original formulation of atovaquone (750 mg tablets four times a day) as a single anti-
toxoplasmic agent was reported to prevent relapse in 48 of 65 (76%) AIDS patients with mean
CD4 counts of 29/µl . More recently, a new formulation of atovaquone (1500 mg suspension)
in combination with pyrimethamine or sulfadiazine was reported to prevent relapse in 19 of 20
We have previously shown the efficacy of atovaquone nanosuspensions in the “acute” i.v.
treatment of TE in a murine model . To investigate the therapeutic efficacy of atovaquone for
oral “maintenance” therapy, we expanded the murine model of reactivated TE in mice deficient
in the interferon regulatory factor 8 (ICSBP/IRF-8)  by adding a phase of oral “maintenance”
treatment after the course of acute i.v.drug.
Results of the present study reveal that atovaquone maintenance therapy in doses equivalent to
the application in humans protected mice against reactivated TE and death. Atovaquone-treated
mice did not develop signs of inflammation in the brain parenchyma nor in the meninges.
Atovaquone maintenance therapy was superior to standard therapy with pyrimethamine plus
sulfadiazine for secondary prophylaxis of TE.
MATERIALS AND METHODS
Cysts of the ME49 strain of T. gondii
were obtained from brains of NMRI-mice
that had been infected intraperitoneally with 10 cysts 2-4 months before. Mice were sacrificed by
asphyxiation with CO2, their brains 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
Mice and infection.
Inbred female ICSBP/IRF-8-/- mice on C57BL/6-background were bred
and maintained under special pathogen-free conditions in the animal facility of the Institute for
Infection Medicine, Charité Campus Benjamin Franklin, Berlin. 8-to-12-weeks-old ICSBP/IRF-8-
/- mice were orally infected. Mice were treated with sulfadiazine (Sigma-Aldrich, Deisenhofen,
Germany) in drinking water (400 mg/l) 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 days after discontinuation of acute
i.v. atovaquone therapy- daily treatment with antiparasitic drugs as maintenance therapy was
initiated p.o. for 1 week. At day 16 – the time point when control mice showed symptoms of
disease and/ or began to succumb – their brains, livers, lungs and serum 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 4 to 6 mice in each experimental group to
study mortality and histological changes. HPLC and MS analysis were performed on organs and
serum samples of 3 to 4 mice per group. Experiments were repeated at least three times.
naphthoquinone was obtained from Glaxo-Smithkline (München, Germany). Atovaquone
nanosuspensions (ANS) 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 of 1% Tween 80 (ICI Surfactants, Eversberg, Belgium), using an
Ultra turrax T 25 (Janke and Kunkel, Staufen, D). The coarse pre-dispersion 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 1500 bar (20 cycles) . Particles
were preserved by thiomersal (Sigma-Aldrich) at a concentration of 0.001% (wt/ vol). Iso-
osmolarity was achieved by adjusting with glycerole (Sigma-Aldrich) at a concentration of 2.25%
(wt/ vol). Particle size and width of distribution (polydispersity index) were determined by
photon correlation spectroscopy (PCS) (Malvern Zetasizer 4, Malvern Instruments, Malvern,
U.K.) and laser diffractometry using a Coulter LS 230 (Beckman-Coulter, Krefeld, Germany).
Mean diameter measured by PCS was 459 ± 2 nm with a polydispersity index of 0.29 ± 0.02,
99% (vol/ vol) of particles had a diameter of below 2.155 µm (LD99). Atovaquone suspension
(Wellvone®, Glaxo-Smithkline) for oral maintenance therapy was diluted in PBS. Mice received
100, 50, or 25mg/kg body weight in a total volume of 0.2 ml by gavage.
Organs were excised and fixed in a solution containing 5% formalin and
0embedded in paraffin. Sagittal sections of brains and cross sections of livers and lungs were
stained with hematoxilin eosin (H&E) according to a standard procedures or by the
immunoperoxidase method with rabbit anti-T. gondii
immunoglobulin G antibody . 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, California, USA) and with the primary antibody rabbit-anti-T.
. For production of rabbit-anti-T.gondii
antibodies, rabbits were orally infected with 10
cysts ME 49, treated with 300 mg/ l sulfadiazine. Sera were harvested after a boost with T. gondii
(RH) 15 days after infection. After rinsing with modified PBS, sections were incubated with
swine-anti-rabbit immunglobulin (1:100) (DAKO). Finally sections were incubated with rabbit
peroxidase anti-peroxidase (1:100) (Sigma-Aldrich) and with diaminobenzidine (DAKO)
development solution after rinsing. Sections stained with H&E were evaluated for inflammatory
changes and sections stained with the immunoperoxidase method were evaluated for the numbers
of T. gondii
cysts and T. gondii
tachyzoites or antigens. Number of inflammatory foci and
parasites were counted under 100x magnification in 3 optical fields. A total of 3 sections of each
organ of 4 mice in each experimental group were evaluated for the numbers of inflammatory foci
and the numbers of T. gondii
cysts, tachyzoites, and antigens. A score was developed modified
after the score described by Araujo et al. . Briefly, normal brain was scored 1. A score of 2
reflected mild meningeal and parenchymal mononuclear cell inflammation, a score of 3 severe
meningeal and parenchymal inflammation, and a score of 4 severe meningeal inflammation plus
severe parenchymal inflammation with necrosis. The score is given as mean ± SD for at least 4
High performance liquid chromatography.
Weighted tissue samples of serum (200 µl),
liver and lung (50-300 mg) were homogenized in 5 ml extraction solution consisting of 2% (vol/
vol) isoamyl alcohol and 98% (vol/ vol) hexane in a glass-teflon homogenizer . 0.1 ml serum
was diluted in 5 ml extraction solution. After adding of 1 ml phosphate buffer samples were spun
for 20 min in a rotating mixer . Suspensions were centrifuged for 10 min at 2800 g in a
temperature controlled centrifuge at 10 °C. Four ml supernatant were evaporated to dryness in a
rotating vacuum centrifuge. The dry residue was re-dissolved in mobile phase (aqueous solution
of 50% (vol/ vol) acetonitrile and 5% methanol (vol/ vol), pH 2.65) [17-19]. The samples were
chromatographed on a reversed-phase column (Spherisorb C1, Waters, USA) guarded with a C
18 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 using computer software (SQS 98,
Perkin Elmer). Detection limit of this method was 0.6 mg/ l serum. Limits of quantitation for
tissues were approximately 0.5 mg/ kg tissue. Inter-assay precision for serum (c.v.) 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)
Since atovaquone concentrations were low in brains of mice treated with
atovaquone maintenance therapy, we used mass spectrometry to quantitate atovaquone in brains
of mice. 25µl aliquots of serum were mixed with 100µl 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 13000g for 6 min.
The supernatants were transferred to clean tubes and aliquots of 20µl were injected onto a
reversed-phase C18 column (Eurospher; 5µ; 4,6mm x 30mm; Knauer, Berlin, Germany). The
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 the following components: Rheos 2000 HPLC pump (Flux
Instruments®, Basel, Switzerland) and a 233 XL autosampler (Gilson Abimed®). HPLC
separation was achieved with mobile phase gradient elution (flow 1,5 ml/min) using the
following sequence: 0 min: 100% A; – 0,1 min: 25% A; – 3,0 min: 25% A; - 3,1 min: 100% A;
The majority (80%) of the effluent was split off before entering the MS.
An API 3000 mass spectrometer (Applied Biosystems®,California,USA) equipped with an ESI
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);
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 8-
point standard calibration curve was analyzed; atovaquone concentrations ranged from 0.617
For quantification of atovaquone in brain tissue the organs were weighed into polypropylene
microreaction vials and homogenized mechanically using a pestle. After addition of acetonitrile
(5µl/mg brain tissue) samples were vigorously vortexed and sonicated for 60 minutes. The
suspended brain tissue was sedimented at 13000 g for 6 minutes and 250 µl aliquots of the
supernatant (containing the extract of 50 mg of brain tissue) were transferred into clean
polypropylene vials and 20 µl of the internal standard solution (mycophenolat 0,3 mg/ml) 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
Fisher’s exact test was used to compare survival rates. Differences in
numbers of inflammatory foci and parasite numbers were analyzed using the Student`s t
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 (10mg/kg body weight) do not develop reactivated
toxoplasmosis whereas all control mice died within 2 weeks after withdrawal of sulfadiazine .
To determine the maximum duration between i.v. injections of atovaquone nanosuspensions
treatment of mice with 10 mg/kg body weight of atovaquone every other day or every third day
was compared (Tab. 1). Both treatment regimens showed equal therapeutic efficacy in the murine
model of reactivated toxoplasmosis. Whereas all control mice died, mice treated with 10 mg/kg
atovaquone every second or third day survived the infection (Tab. 1). Treated mice did not
develop parasite-associated inflammatory changes in their brains (Tab.1). The optimal dose of
i.v. atovaquone for treatment of acute reactivated toxoplasmosis was determined by comparing
treatment with 10, 5, and 2.5 mg atovaquone nanosuspension/kg body weight. Whereas mice
treated with 10 mg atovaquone nanosuspensions did not develop parasite-associated
inflammatory changes and survived the infection, 20% and 45% of mice treated with a dose of
either 5 or 2.5 mg atovaquone nanosuspensions, respectively developed parasite-associated
inflammatory changes in their brains and died. High concentrations of atovaquone were only
detectable by HPLC in serum and organs of mice treated with 10 mg/kg body weight (Tab. 1). In
contrast, mice treated with 5 mg/kg showed low atovaquone concentrations in serum and liver
whereas atovaquone was undetectable by HPLC in brains. Therefore, 10 mg/kg atovaquone
nanosuspensions administered every third day, for the i.v. treatment of acute reactivated
toxoplasmosis were used in all experiments to investigate the therapeutic efficacy of a variety of
antiparasitic drugs for subsequent maintenance therapy.
Murine model of reactivated toxoplasmic encephalitis for the evaluation of
. 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 withdrawal of sulfadiazine
which was used to establish latent infection in the immunocompromized mice . Two days
thereafter when mice reactivated the infection, acute therapy with atovaquone nanosuspensions
was initiated every third day at a dose of 10 mg/kg body weight (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). 16 days after
discontinuation of sulfadiazine, serum and organs were obtained and mortality of mice was
monitored in a separate group of mice.
Effect of atovaquone maintenance therapy on survival of mice with reactivated
. 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 Patinets) 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 towards 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 toxoplasmic encephalitis (Fig. 2).
Effect of atovaquone maintenance therapy on histological changes in mice with
reactivated toxoplasmic encephalitis
. Histological findings in brains and livers obtained on day
7 after initiation of maintenance therapy (time point when maintenance therapy was stopped)
paralleled the results of survival as shown above (Tab. 2, 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 livers of control mice, we
detected numerous areas of inflammation associated with parasites (Tab. 2). In contrast,
atovaquone maintenance therapy prevented the development of toxoplasmic encephalitis; neither
mice treated with 100 mg/kg body weight nor those treated with 50 mg/kg body weight showed
any signs of inflammation in their brains (Fig. 3B,C) or livers. Also parasites were undetectable
in either organ. Similar results were obtained in mice treated with pyrimethamine plus
sulfadiazine (Fig. 3D). However sings of inflammation were observed In contrast, 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. A decrease in inflammation both in the meninges and the brain parenchyma was
noted in mice treated with trovafloxacin (Fig. 3F).
Atovaquone concentrations in serum and organs.
To determine atovaquone
concentrations, serum, brains, lungs, and livers were obtained on day 8 after initiation of
maintenance therapy. Atovaquone concentrations were determined in serum samples by HPLC
and mass spectrometry 24h after the last administration of drug; mice orally treated with 50 or
100 mg/kg atovaquone showed high drug concentrations of 15.00 mg/l or higher in their sera
(Fig. 4A). Atovaquone was detectable in livers and lungs at lower concentrations (Fig. 4B).
Atovaquone concentrations in brains of mice treated with either 50 or 100 mg/kg were 0.22 ±
0.05 mg/kg and 0.34 ± 0.14 mg/kg, respectively (Fig. 4B).
Results of the present study reveal that mice treated orally with atovaquone maintenance therapy
in doses equivalent to the application in humans did not develop reactivation TE.
These results were obtained in a new murine model of reactivated toxoplasmosis that closely
mimics signs of reactivated toxoplasmosis in immunocompromized patients, including the
presence of parasite-associated focal necrotic lesions in the brain parenchyma and meningeal
inflammation.This pathological changes resulted in death of mice.
In the past studies on the efficacy of antiparasitic drugs for maintenance therapy have been
performed using in vitro systems and/or different animal models [7, 8, 20-22] . However, none of
these model systems represented reactivation of latent toxoplasmosis and antiparasitic drugs for
maintenance therapy were never administrated orally following a course of acute therapy. Thus,
the murine model described in the present study for the first time allowed to adaequately study
the efficacy of antiparasitic drugs for maintenance therapy.
The current recommendations for maintenance therapy against TE in patients with AIDS
include as first choice the use of sulfadiazine (500 – 1000 mg orally four times daily) plus
pyrimethamine (25 – 50 mg orally daily); this recommendation is based on the strong evidence
for efficacy and clinical benefit observed in randomized clinical trials (A1 strength of
recommendation by the US Public Health Service and the Infectious Disease Society of America)
. Alternatively, clindamycin (300 – 450 mg orally every 6 – 8 hours) plus pyrimethamine (25
– 50 mg by mouth daily) or atovaquone (750 mg orally every 6 – 12 hours) with or without
pyrimethamine (25 – 50 mg orally daily) may be given; the evidence for efficacy of these
alternative treatments is based on clinical experience, descriptive studies, or reports of consulting
committees (CIII strength of recommendation) insufficient to support recommendation . In a
study by Katlama et al. , 65 AIDS patients intolerant of standard treatment regimens received
atovaquone (750 mg four times daily) as single maintenance therapy. Atovaquone was found to
be efficacious in 74% of patients . The efficacy of 74% observed in humans by
Katlama and coworkers is lower than the efficacy of 100% observed in mice in the present study.
However, whereas 4 x 750 mg of the old tablet form of atovaquone were administered to patients,
mice received the new suspension formulation of atovaquone in equivocal dosis. It has been
shown, that Hifat (toast with 56g butter) increased the uptake of atovaquone tablets by a factor of
3.9 (AUC) and 5.6 (Cmax) . Atovaquone aqueous suspension or oily solution in miglyol also
increased the AUC and Cmax by a factor of 1.7 and 2.4, respectively. We therefore
hypothesize that the current formulation of atovaquone (oral suspension with Xanthan Gum and
Poloxamer 188, Wellvone® package insert 1997) should prove markedly more efficacious than
the old formulation for maintenance therapy against reactivation of TE in humans. In this respect,
the combination of atovaquone suspension plus either pyrimethamine or sulfadiazine was
effective as maintenance therapy in 19 (95%) of 20 patients with AIDS Chirgwin et al. .
However, the study did not allow to determine whether the new atovaquone formulation or rather
the combination therapy mediated the protective effects.
In all regimens including sulfadiazine or pyrimethamine, leucovorin therapy must be
added to prevent bone marrow suppression. In addition, sulfadiazine as well as clindamycin
therapy are hampered by allergic reactions which are observed in approximately 30% of patients;
between 11 and 30% of patients discontinue maintenance therapy . In contrast, atovaquone
maintenance therapy has been reported to be very well tolerated . Only two (3.0%) of 65
AIDS patients experienced gastrointestinal side effects including nausea and vomiting and
therefore had to discontinue atovaquone maintenance therapy .
We compared the therapeutic effect oral administration of atovaquone in dosages between 25
and 100 mg/kg body weight. Atovaquone dosages of 100 and 50 mg/kg body weight were
efficacious for maintenance therapy against TE whereas atovaquone at a dose of 25 mg/kg body
weight did not protect mice from reactivated toxoplasmosis (data not shown). Dosage of 50 and
100 mg/kg body weight resulted in serum levels of 14.5 and 20.8 mg/l, respectively; serum levels
achieved in infected mice thus fall above the MIC for T. gondii
reported by us and others to be in
the nanogramm range [7-9] The dosage of 50 mg/kg body weight that proved efficacious in the
murine model of reactivated TE is equivocal to the dosage reported efficacious in humans (750
mg 4 times daily) by Katlama et al. . Since we were interested in brain concentrations of
atovaquone, a mass spectrometry assay for the detection of atovaquone was established.
Concentrations achieved in brains of infected mice (0.22 – 0.34 µg/mg) also fell above the MIC
of 0,01 µg/ml for T. gondii
In the past, acute therapy for TE has been investigated in murine models using drug-induced
immunosuppression . In mice latently infected with the cystogenic ME49 strain, reactivation
of TE was induced by a 2-week course of dexamethasone. Administration of atovaquone plus
clindamycin (dosages of 50 mg/kg body weight each or higher) significantly prolonged survival
of mice and significantly reduced the numbers of brain cysts . A similar effect of atovaquone
on cyst numbers was also reported by Araujo et al.  in vitro and in a murine model of chronic
progressive toxoplasmosis in susceptible CBA/ca mice.
In conclusion, the murine model of reactivated toxopasmosis described in the present study,
proved valuable to study efficacy of antiparasitic drugs for maintenance therapy against TE. The
therapeutic effect of Atovaquone should be further evaluated a clinical trials.
We thank Berit Söhl Kielczynski, Sigrid Ziesch, Andrea Maletz, Solvy Wolke for expert
technical assistance and Helmut Hahn for continuous support.
This work was supported by the Forschergruppe 463, German Research Foundation (DFG). Parts
of this work were realized within the Competence Network on HIV/AIDS (German Ministry of
Education and Research, BMBF, grant 01KI0211).
Cook, A. J., Gilbert, R. E., Buffolano, W., Zufferey, J., Petersen, E., Jenum, P. A.,
Foulon, W., Semprini, A. E. and Dunn, D. T.,
Sources of toxoplasma infection in
pregnant women: European multicentre case-control study. European Research Network
on Congenital Toxoplasmosis. Bmj
Montoya, J. G. and Liesenfeld, O.,
Luft, B. J. and Remington, J. S.,
Toxoplasmic Encephalitis in AIDS (AIDS
Commentary). Clinical Infectious Diseases
Porter, S. B. and Sande, M.,
Toxoplasmosis of the central nervous system in the
Acquired Immunodeficiency Syndrome. N Engl J Med
Liesenfeld, O., Wong, S. Y. and Remington, J. S.,
Toxoplasmosis in the Setting of
AIDS. In Bartlett, J. G., Merigan, T. C. and Bolognesi, D.
(Eds.) Textbook of AIDS
, 2nd Edition Edn. Williams & Wilkins, Baltimore 1999, pp 225-259.
Katlama, C., De Wit, S., O'Doherty, E., Van Glabeke, M. and Clumeck, N.,
Pyrimethamine-clindamycin vs. pyrimethamine-sulfadiazine as acute and long-term
therapy for toxoplasmic encephalitis in patients with aids. Clin Infect Dis
Araujo, F. G., Huskinson, J. and Remington, J. S.,
Remarkable in vitro
and in vivo
activities of the hydroxynaphthoquinone 566C80 against tachyzoites and tissue cysts ofToxoplasma gondii
. Antimicrob. Agents Chemother.
Baggish, A. L. and Hill, D. R.,
Antiparasitic agent atovaquone. Antimicrob Agents
Scholer, N., Krause, K., Kayser, O., Muller, R. H., Borner, K., Hahn, H. and
Atovaquone nanosuspensions show excellent therapeutic effect in a new
murine model of reactivated toxoplasmosis. Antimicrob Agents Chemother
Kovacs, J. A.,
Efficacy of atovaquone in treatment of toxoplasmosis in patients with
AIDS. The NIAID-Clinical Center Intramural AIDS Program. Lancet
Chirgwin, K., Hafner, R., Leport, C., Remington, J., Andersen, J., Bosler, E. M.,
Roque, C., Rajicic, N., McAuliffe, V., Morlat, P., Jayaweera, D. T., Vilde, J. L. and
Luft, B. J.,
Randomized phase II trial of atovaquone with pyrimethamine or sulfadiazine
for treatment of toxoplasmic encephalitis in patients with acquired immunodeficiency
syndrome: ACTG 237/ANRS 039 Study. AIDS Clinical Trials Group 237/Agence
Nationale de Recherche sur le SIDA, Essai 039. Clin Infect Dis
Katlama, C., Mouthon, B., Gourdon, D., Lapierre, D. and Rousseau, F.,
as long-term suppressive therapy for toxoplasmic encephalitis in patients with AIDS and
multiple drug intolerance. Atovaquone Expanded Access Group. Aids
Holtschke, T., Lohler, J., Kanno, Y., Fehr, T., Giese, N., Rosenbauer, F., Lou, J.,
Knobeloch, K. P., Gabriele, L., Waring, J. F., Bachmann, M. F., Zinkernagel, R. M.,
Morse, H. C., 3rd, Ozato, K. and Horak, I.,
Immunodeficiency and chronic
myelogenous leukemia-like syndrome in mice with a targeted mutation of the ICSBP
Grau, M. J., Kayser, O. and Muller, R. H.,
Nanosuspensions of poorly soluble drugs--
reproducibility of small scale production. Int J Pharm
Conley, F. K., Jenkins, K. A. and Remington, J. S., Toxoplasma gondii
infection of the
central nervous system. Use of the peroxidase-antiperoxidase method to demonstrate
toxoplasma in formalin fixed, paraffin embedded tissue sections. Hum. Pathol.
Araujo, F. G., Suzuki, Y. and Remington, J. S.,
Use of rifabutin in combination with
atovaquone, clindamycin, pyrimethamine, or sulfadiazine for treatment of toxoplasmic
encephalitis in mice. Eur J Clin Microbiol Infect Dis
Hannan, S. L., Ridout, G. A. and Jones, A. E.,
Determination of the potent
antiprotozoal compound atovaquone in plasma using liquid-liquid extraction followed by
reversed-phase high-performance liquid chromatography with ultraviolet detection. J
Chromatogr B Biomed Appl
Hansson, A. G., Mitchell, S., Jatlow, P. and Rainey, P. M.,
liquid chromatographic assay for atovaquone. J Chromatogr B Biomed Appl
DeAngelis, D. V., Long, J. D., Kanics, L. L. and Woolley, J. L.,
liquid chromatographic assay for the measurement of atovaquone in plasma. J
Djurkovic-Djakovic, O., Nikolic, T., Robert-Gangneux, F., Bobic, B. and Nikolic, A.,
Synergistic effect of clindamycin and atovaquone in acute murine toxoplasmosis.Antimicrob Agents Chemother
Romand, S., Pudney, M. and Derouin, F.,
In vitro and in vivo activities of the
hydroxynaphthoquinone atovaquone alone or combined with pyrimethamine,
sulfadiazine, clarithromycin, or minocycline against Toxoplasma gondii. Antimicrob
Brun-Pascaud, M., Rajagopalan-Levasseur, P., Chau, F., Bertrand, G., Garry, L.,
Derouin, F. and Girard, P. M.,
Drug evaluation of concurrent Pneumocystis carinii,
Toxoplasma gondii, and Mycobacterium avium complex infections in a rat model.Antimicrob Agents Chemother
Kaplan, J. E., Masur, H. and Holmes, K. K.,
Guidelines for preventing opportunistic
infections among HIV-infected persons--2002. Recommendations of the U.S. Public
Health Service and the Infectious Diseases Society of America. MMWR Recomm Rep
Rolan, P. E., Mercer, A. J., Weatherley, B. C., Holdich, T., Meire, H., Peck, R. W.,
Ridout, G. and Posner, J.,
Examination of some factors responsible for a food-induced
increase in absorption of atovaquone. Br J Clin Pharmacol
Djurkovic-Djakovic, O., Milenkovic, V., Nikolic, A., Bobic, B. and Grujic, J.,
Efficacy of atovaquone combined with clindamycin against murine infection with a
cystogenic (Me49) strain of Toxoplasma gondii. J Antimicrob Chemother
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
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 5 mice were used in each group.
Results shown are pooled from two independent experiments.
FIG. 3. Histological changes in brains of mice with reactivated toxoplasmic encephalitis
at day 8 after initiation of maintenance therapy. Small arrows indicate parasitic foci
(parasitophorus vacuoles and parasitic antigen), large arrows indicate inflammatory foci.
Immunoperoxidase staining, magnification, x100. (A) control mice, atovaquone-treated mice (B)
(100mg/kg), (C) (50mg/kg),(D) pyrimethamin plus sulfadiazine-treated mice,(E) pyrimethamin
plus clindamicin-treated mice,(F) trovafloxacine-treated mice and (G) sufadiazine-treated mice.
Sections shown are representative for at least 4 mice per group; experiments were repeated 3
FIG. 4. Concentrations of atovaquone in serum (A), brains, livers, and lungs (B) of mice
with reactivated toxoplasmic encephalitis. Mice were treated with indicated concentrations of
atovaquone and killed 7 days after initiation of oral atovaquone maintenance therapy (1 day after
thenlast dose of atovaquone). Atovaquone concentrations were determined by mass spectrometry
(serum, brains) and HPLC (livers, lungs). Values are derived from at least 3 mice per group and
are representative for 2 experiments performed.
Adequate intake of Vitamin D and calcium The National Osteoporosis Foundation advises al individuals to obtain an adequate intake of dietary calcium (at least 1200 mg per day, including supplements if necessary) and vitamin D (400 to 800 IU per day for individuals at risk of deficiency). An adequate intake of calcium is necessary for the acquisition of peak bone mass and maintenance of bone h
THE WEST BENGAL NATIONAL UNIVERSITY OF JURIDICAL SCIENCES ALTERNATE TO ALTERNATIVES CRITICAL REVIEW OF THE CLAIMS OF ADR Jasmine Joseph Assistant Professor NUJS Working Paper Series NUJS/WP/2011/01 The NUJS Working papers are research in progress carried out in NUJS and are aimed at disseminating the preliminary findings and arguments on an ongoing research for the