Toxoplasmosis Research Laboratory, Institute for Medical Research, PO Box 102, 11129 Belgrade, Yugoslavia
Received 21 January 2002; returned 13 May 2002; revised 21 June 2002; accepted 16 September 2002
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Abstract |
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Introduction |
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The hydroxynaphthoquinone atovaquone (ATO) appears to be active not only against tachyzoites, but also against bradyzoite-containing T. gondii cysts.5 Mitochondrial cytochrome b has recently been identified as the target for ATO in T. gondii.6 ATO has been shown to be effective in animal models of infection, alone710 or combined with other drugs such as pyrimethamine and sulfadiazine,11 or rifabutin,4,12 and is being used increasingly in human therapy.1315 We have reported on the synergic activity of ATO and clindamycin (CLI), a widely used alternative drug16,17 with well-established anti-T. gondii activity,1821 in murine toxoplasmosis induced with the mouse-virulent (type-1) RH strain of T. gondii.22 However, since type-2 strains account for 65% of cases of reactivation of toxoplasmosis in AIDS,23 in this report we present data on the anti-T. gondii efficacy of ATO and CLI in murine infection induced with Me49 as a typical type-2 strain, including a model of T. gondii recrudescence.
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Materials and methods |
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Female Swiss-Webster mice (Medical Military Academy Animal Research Facility, Belgrade, Yugoslavia), 6 weeks old, weighing 1822 g at the beginning of experiments, were used. Mice were housed five or six per cage (depending on the experiment) and offered drinking water and regular mouse feed ad libitum, unless otherwise specified. All animal studies were performed with the approval of a local (Institute for Medical Research) ethics committee.
Parasites
Brain cysts of the Me49 strain of T. gondii (initially provided by Dr J. P. Dubey, Beltsville, MD, USA), regularly maintained by passage through Swiss-Webster mice twice a year, were used to establish chronic infection. To obtain cysts for experimental infections, mice infected at least 8 weeks previously were killed by asphyxiation in chloroform and their brains removed and homogenized in a Teflon homogenizer with 1 mL of saline each. For cyst enumeration, 25 µL of the brain suspensions was placed on slides and microscopically counted. The number of cysts per brain was calculated by multiplying the number counted in four drops by 10, giving a threshold sensitivity of 10 cysts per brain. For experimental infections, fresh mice were inoculated by intraoesophageal gavage with 250 µL brain suspensions assessed to contain 10 or 20 cysts. In our experience, inoculation of 10 cysts produces consistent infection without mortality, whereas inoculation of 20 cysts results in mortality;24 thus, to allow for differences in survival after early drug treatment, inocula of 20 cysts were used to develop acute infection, whereas 10 cysts were inoculated to establish chronic infection.
Drugs
ATO (micronized powder, lot 291604A; GlaxoWellcome, Stevenage, UK, kindly provided by Dr Simon Lister) was administered at 5, 25, 50 and 100 mg/kg body weight per day.
CLI (CLI hydrochloride powder, lot 353YS; Upjohn, supplied by Yusapharm, Belgrade, Yugoslavia) was administered at 25, 50 and 400 mg/kg/day.
On the basis of the observation that mice consume 4 g of food per day,18,20 the desired dosages were obtained by adding ATO 0.025, 0.125, 0.25 and 0.5 mg and CLI 0.125, 0.25 and 2.0 mg, respectively, per 1 g of ground mouse feed. Fresh food was supplied daily.
These dosages were selected on the basis of previous work7,12,18,19,21 as effective (ATO 100 and CLI 400 mg/kg/day) or, to better reveal the effects of the combined therapy, as suboptimal (all lower dosages of both drugs).
To control for drug side-effects, in a preliminary experiment, separate groups of mice were given ATO (100 mg/kg/day) and CLI (400 mg/kg/day) for 3 weeks and ATO+CLI (100+400 mg/kg/day) for 2 weeks. No clinically significant toxicity (ruffled fur, lethargy, weight loss) was observed over a period of 3 months.
Dexamethasone-sodium-phosphate (DXM; ICN Galenika, Belgrade, Yugoslavia) was given at a dosage of 2.5 mg/kg/day per mouse, obtained by dissolving 5 mg of DXM per 1 L of drinking water. Treated and untreated water was changed three times a week.
Hydrocortisone-21-acetate (CA, lot. 87494; ICN Biomedicals, Aurora, OH, USA) was administered at 50 mg/kg by subcutaneous injection three times a week.
Co-amoxiclav (Panklav; Panfarma, Belgrade, Yugoslavia) was given in drinking water at 1 g/L for the period of administration of DXM and CA to prevent bacterial superinfection.
Experimental design
Acute infection model. Two separate experiments were performed. Groups of mice (n = 1520) inoculated with 20 cysts were arbitrarily assigned to treatment with ATO and CLI, as follows: ATO 100 and CLI 400 mg/kg/day, alone and combined (experiment 1); ATO 25 and CLI 50 mg/kg/day, alone and combined (experiment 2); or left untreated to serve as controls. In both experiments, treatment was initiated day 4 post-infection and continued for 14 consecutive days. Mice were observed for 30 days after the end of treatment, at which point the survivors were killed and the brains removed and cysts counted as described above.
Chronic infection model. Groups of mice (n = 15) inoculated with 10 cysts 3 months previously were arbitrarily assigned to treatment with ATO 100, CLI 400 and ATO+CLI 100+400 mg/kg/day for 14 consecutive days or left untreated as controls. Fourteen days after the end of treatment, they were killed and processed as above.
Model of chronic infection in the immunosuppressed host (reactivated toxoplasmosis). DXM and CA were used to induce immunosuppression in chronically infected mice. The choice of corticoid drug regimens was based on our previous work,24 which showed that DXM, but not CA alone, induces immunosuppression leading to recrudescence of T. gondii in previously latent infection. However, the addition of CA to DXM potentiates the immunosuppressive effect of DXM. Clinically manifest disease followed by mortality begins after 2 weeks and continues throughout the period of corticoid administration. Thus, in experiment 1, groups of mice (n = 12) inoculated with 10 cysts 3 months previously were arbitrarily given DXM alone or combined with CA for 3 weeks and then treated with ATO+CLI 5+25 and 50+50 mg/kg/day for the next 4 weeks. Immediately after the end of treatment, the brain cysts were harvested from the survivors. In experiment 2, groups of chronically infected mice (n = 18) were pre-treated with DXM for 14 days and then arbitrarily given ATO+CLI 25+50, 50+50 and 100+400 mg/kg/day for 3 weeks. Five mice from each treatment group were killed immediately at the end of treatment and examined for brain cyst burdens, whereas the remaining mice were observed for the next 12 weeks, at which time (day 120 after initiation of immunosuppressive treatment) the survivors were killed and processed as above.
In all models, mice were monitored daily and deaths were recorded. In mice dying during the experiments as well as those surviving the observation period as specified in the respective model, brain parasite burdens were determined as described above.
Bioassay
If no cysts were observed in at least four samples of a brain tissue preparation, the remainder of the brain homogenate was inoculated by mouth into two fresh mice per sample (450 µL each). Mice were killed after 6 weeks and brain tissue examined for the presence of brain cysts.
Statistical analysis
Survival rates in particular groups were estimated by the KaplanMeier product limit method and compared by the log rank (two curves) and multiple sample (three or more curves) tests. Differences in the numbers of cysts between groups were examined by Students t-test. The level of statistical significance was P = 0.05.
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Results |
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The effects of a 2 week course of treatment with ATO and CLI initiated day 4 post-infection were monitored by 7 week survival, as well as by the brain cyst burdens at the time of death or by the end of the observation period. Compared with the survival in untreated mice of 60% and 65%, respectively, in experiments 1 and 2 all treated mice survived significantly better. Treatment with the lowest dosages of the drugs given alone (ATO 25 and CLI 50 mg/kg/day) protected 93% (P = 0.043 and 0.047, respectively) of mice, whereas the higher dosages of each drug alone (ATO 100 and CLI 400 mg/kg/day), as well as both combinations (ATO+CLI 25+50 and 100+400 mg/kg/day), afforded protection to virtually all mice (P = 0.003).
Moreover, the treatment clearly reduced the number of cysts (Figure 1). However, important differences between the effects of ATO and CLI were observed. Although, compared with untreated mice, the cyst burdens were reduced in mice treated with CLI, the difference reached significance (P = 0.027) only in experiment 2. On the other hand, in both experiments, the cyst burdens in mice treated with ATO both alone and combined with CLI were significantly lower than in both untreated mice (P < 0.0001) and mice treated with CLI alone (P = 0.0002 and 0.0008, respectively, in experiment 1; and P = 0.0044 and 0.005, respectively, in experiment 2). However, there were no differences in either experiment between the effects of ATO both alone and combined with CLI (P > 0.05).
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Chronic infection model
In chronically infected mice, which all survived throughout the experiment whether treated or not, 2 weeks after the end of a 2 week course of treatment with ATO 100 and CLI 400 mg/kg/day alone, the cyst burdens were insignificantly (P > 0.05) decreased, i.e. to 75.9% and 76.4%, respectively, of those in untreated mice (Figure 2). However, compared with untreated mice, treatment with ATO combined with CLI reduced the cyst burden significantly (P = 0.033). Compared with mice treated with ATO and CLI alone, the cyst burden in mice treated with the combined drugs was lowered to only 43%; although this difference did not reach significance, it came close (P = 0.055 and 0.078, respectively).
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In experiment 1, chronically infected mice pre-treated with DXM alone or combined with CA for 3 weeks were submitted to a 4 week course of treatment with combinations of ATO+CLI 5+25 and 50+50 mg/kg/day. All untreated mice immunosuppressed with both DXM and CA succumbed to the infection by day 43, whereas 18% of those suppressed with DXM alone survived the observation period (Figure 3). Compared with these, mice treated with ATO+CLI 5+25 mg/kg/day did not survive any better (0% and 18% survival, respectively, for the DXM+CA and DXM pre-treated groups). In contrast, treatment at 50+50 mg/kg/day enhanced the survival of mice on suppressive regimen to 30%. However, the expected probabilities of survival did not vary significantly either among all groups taken together (P = 0.2919, multiple sample test) or between any two groups (P > 0.05). Similarly, although the cyst burdens (in all mice dying during the treatment/observation period or survivors killed at the end of the experiment) (Figure 4) were decreased, compared with untreated mice on either suppressive regimen, in mice treated with ATO+CLI 5+25 mg/kg/day by two-fold (to 59.2% and 46.4% of the control values) and even by four-fold in mice treated with ATO+CLI 50+50 mg/kg/day (to 27.2% and 23.1% of the control values), these differences were not significant, obviously due to high standard deviations in the untreated mice. Still, the cyst reduction induced by the higher dosage combination (50+50 mg/kg/day) in mice on either suppressive regimen was close to significant compared with the respective controls (DXM+CA, P = 0.07; DXM, P = 0.058).
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Discussion |
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In acutely infected mice, however, treatment with combinations of ATO and CLI, in addition to protecting 100% of the mice, significantly reduced the cyst burden compared with treatment with CLI alone, but not with ATO alone, indicating that in this model the most effective component of the combination was ATO. ATO both alone and combined with CLI greatly reduced the brain cyst burden, which remained extremely low weeks after treatment, even below 10 in some, as shown by their apparent absence on direct examination. However, positive bioassays in all such cases indicate that the drugs appear, at least under the experimental conditions used (duration of treatment, drug dosages), incapable of eradicating the parasite.
On the other hand, in chronically infected mice, ATO combined with CLI significantly reduced the brain cyst load compared not only with no treatment but also with either drug alone. A stronger effect of the combined drugs as opposed to a similarly insignificant reduction of the parasite burden by each drug alone, in view of the known activity of ATO against cysts, probably reflects the natural history of infection with the Me49 strain; this strain tends continuously to give rise to new cyst formation, presumably preceded by cyst rupture and proliferation of tachyzoites that are then converted into bradyzoites; CLI is active against these tachyzoites, reducing their number and hence reducing the number of newly formed cysts.
The discrepancy between the effect of ATO in acute infection, where it reduced the brain cyst burden comparably to the combined drugs, and its lower efficacy in 3-month-old chronic infections, may be explained by ATO being more effective against metabolically active immature bradyzoites than against mature ones.8
Most importantly, ATO combined with CLI showed activity in models of corticoid drug-induced recrudescence of T. gondii, as shown by significantly increased brain cyst burdens in immunosuppressed mice. This activity was dependent directly on the drug dosage and indirectly on the level of immunosuppression. In immunosuppression induced by pre-treatment with both DXM and CA, survival was lower and brain cyst burdens higher than in mice suppressed with DXM alone. The lowest dosages of the drugs required to achieve a protective effect were ATO 25 and CLI 50 mg/kg/day, which is similar to what we had shown in infection induced with the RH strain of T. gondii.22 Duration of corticoid drug administration constituted an important variable of the immunosuppressive protocol. Thus, in mice pre-treated with DXM for 2 and 3 weeks, courses of treatment with ATO+CLI 50+50 mg/kg/day protected 67% and 30%, respectively. Therefore, treatment was more likely to reduce the parasite load below critical levels when initiated after 2 rather than 3 weeks of DXM administration. This is consistent with the reported DXM-induced release of parasites from pre-existing cysts and new cyst formation as early as after 612 days of administration.25 Another important observation was that, when given at effective dosages, the effects of combinations of ATO and CLI were long-term, as shown by the tendency of the brain cyst loads, which increase over time in untreated mice, to continue decreasing for months after the end of treatment. Without attempting to speculate on the underlying reasons, this finding may be of the greatest significance from a clinical standpoint.
Even several-fold lower cyst burdens were not always statistically significant, due, as mentioned, to the wide standard deviations, which probably reflect the variations in the number of bradyzoites within initial cysts (between a few and several hundred),26 and subsequent variability in new cyst formation.
In conclusion, the demonstrated efficacy of ATO combined with CLI in murine infection with a cystogenic strain of T. gondii, taken together with our previous findings of synergic activity of the same drug combination against infection induced with the RH strain, clearly shows the anti-T. gondii potential of this drug combination. In as much as findings in animal models may be extrapolated to the human situation, given that ATO is generally well tolerated13,14 and, moreover, that in combination the drugs may be used at dosages lower than when used alone, which may reduce the rare adverse side-effects of CLI,16,17 the combination of ATO and CLI may offer an efficient yet safe alternative for the treatment of toxoplasmosis in both immunocompetent and immunosuppressed individuals, something that warrants clinical evaluation.
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Acknowledgements |
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Footnotes |
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Present address. Institute for Human Genetics, Am Hubland, Biozentrum, 97074 Wurzburg, Germany.
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References |
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2 . Haverkos, H. W. & the TE Study Group. (1987). Assessment of therapy for toxoplasma encephalitis. American Journal of Medicine 82, 90714.[ISI][Medline]
3 . Leport, C., Raffi, F., Matherson, S., Katlama, C., Regnier, B., Saimot, A. G. et al. (1988). Treatment of central nervous system toxoplasmosis with pyrimethamine/sulfadiazine combination in 35 patients with the acquired immunodeficiency syndrome. Efficacy of long-term continuous therapy. American Journal of Medicine 84, 94100.[ISI][Medline]
4 . Araujo, F. G., Slifer, T. & Remington, J. S. (1994). Rifabutin is active in murine models of toxoplasmosis. Antimicrobial Agents and Chemotherapy 38, 5705.[Abstract]
5 . McCabe, R. E. (2001). Antitoxoplasma chemotherapy. In Toxoplasmosis (Joynson, D. H. M. & Wreghitt, T. G., Eds), pp. 31960. Cambridge University Press, Cambridge, UK.
6 . McFadden, D. C., Tomavo, S., Berry, E. A. & Boothroyd, J. C. (2000). Characterization of cytochrome b from Toxoplasma gondii and Qo domain mutations as a mechanism of atovaquone-resistance. Molecular and Biochemical Parasitology 108, 112.[ISI][Medline]
7 . Araujo, F. G., Huskinson-Mark, J., Gutteridge, W. E. & Remington, J. S. (1992). In vitro and in vivo activities of the hydroxynaphthoquinone 566C80 against the cyst form of Toxoplasma gondii. Antimicrobial Agents and Chemotherapy 36, 32630.[Abstract]
8 . Ferguson, D. J., Huskinson-Mark, J., Araujo, F. G. & Remington J. S. (1994). An ultrastructural study of the effect of the treatment with atovaquone in brains of mice chronically infected with the ME49 strain of Toxoplasma gondii. International Journal of Experimental Pathology 75, 1116.[ISI][Medline]
9 . Gormley, P. D., Pavesio, C. E., Minnasian, D. & Lightman, S. (1998). Effects of drug therapy on Toxoplasma cysts in an animal model of acute and chronic disease. Investigative Ophthalmology and Visual Science 39, 11715.[Abstract]
10 . Sordet, F., Aumjaud, Y., Fessi, H. & Derouin, F. (1998). Assessment of the activity of atovaquone-loaded nanocapsules in the treatment of acute and chronic murine toxoplasmosis. Parasite 5, 2239.[ISI][Medline]
11 . Araujo, F. G., Lin, T. & Remington, J. S. (1993). The activity of atovaquone (566C80) in murine toxoplasmosis is markedly augmented when used in combination with pyrimethamine or sulfadiazine. Journal of Infectious Diseases 167, 4947.[ISI][Medline]
12 . Romand, S., Della Bruna, C., Farinotti, R. & Derouin, F. (1996). In vitro and in vivo effects of rifabutin alone or combined with atovaquone against Toxoplasma gondii. Antimicrobial Agents and Chemotherapy 40, 201520.[Abstract]
13 . Kovacs, J. & the NIAID Clinical Centre Intramural AIDS Program. (1992). Efficacy of atovaquone in treatment of toxoplasmosis in patients with AIDS. Lancet 340, 6378.[ISI][Medline]
14 . Katlama, C., Mouthon, B., Gourdon, D., Lapierre, D., Rousseau, F. & the Atovaquone Expanded Access Group. (1996). Atovaquone as long-term suppressive therapy for toxoplasmic encephalitis in patients with AIDS and multiple drug intolerance. AIDS 10, 110712.[ISI][Medline]
15 . Torres, R. A., Weinberg, W., Stansell, J., Leoung, G., Kovacs, J., Rogers, M. et al. (1997). Atovaquone for salvage treatment and suppression of toxoplasmic encephalitis in patients with AIDS. Atovaquone/Toxoplasmic Encephalitis Study Group. Clinical Infectious Diseases 24, 4229.[ISI][Medline]
16 . Dannemann, B. R., Israelski, D. M. & Remington, J. S. (1988). Treatment of toxoplasmic encephalitis with intravenous clindamycin. Archives of Internal Medicine 148, 247782.[Abstract]
17 . Dannemann, B., McCutchan, J. A., Israelski, D., Antoniskis, D., Leport, C., Luft, B. et al. (1992). Treatment of toxoplasmic encephalitis in patients with AIDS. A randomized trial comparing pyrimethamine plus clindamycin to pyrimethamine plus sulfadiazine. Annals of Internal Medicine 116, 3343.[ISI][Medline]
18 . McMaster, P. R. B., Powers, K. G., Finerty, J. F. & Lunde, M. N. (1973). The effect of two chlorinated lincomycin analogues against acute toxoplasmosis in mice. American Journal of Tropical Medicine and Hygiene 22, 147.[ISI][Medline]
19 . Araujo, F. G. & Remington, J. S. (1974). Effect of clindamycin on acute and chronic toxoplasmosis in mice. Antimicrobial Agents and Chemotherapy 5, 64751.[ISI]
20 . Filice, G. A. & Pomeroy, C. (1991). Effect of clindamycin on pneumonia reactivation of Toxoplasma gondii infection in mice. Antimicrobial Agents and Chemotherapy 35, 7802.[ISI][Medline]
21
.
Vukovi, D., Djurkovi
-Djakovi
, O., Kova
evi
, S., Bobi
, B., Nikoli
, A., Todorovi
, V. et al. (1997). Effect of clindamycin in a murine model of acute toxoplasmosis. Clinical Microbiology and Infection 3, 8994.[Medline]
22
.
Djurkovi-Djakovi
, O., Nikoli
, T., Robert-Gangneux, F., Bobi
, B. & Nikoli
, A. (1999). Synergistic effect of clindamycin and atovaquone in acute murine toxoplasmosis. Antimicrobial Agents and Chemotherapy 43, 22404.
23 . Howe, D. K. & Sibley, L. D. (1995). Toxoplasma gondii is comprised of three clonal lineages: correlation of parasite genotype with human disease. Journal of Infectious Diseases 172, 15616.[ISI][Medline]
24
.
Djurkovi-Djakovi
, O. & Milenkovi
, V. (2001). Murine model of drug-induced reactivation of Toxoplasma gondii. Acta Protozoologica 40, 99106.[ISI]
25 . Odaert, H., Soete, M., Fortier, B., Camus, D. & Dubremetz, J. F. (1996). Stage conversion of Toxoplasma gondii in mouse brain during infection and immunodepression. Parasitology Research 82, 2831.[ISI][Medline]
26 . Dubey, J. P. (1997). Bradyzoite-induced murine toxoplasmosis: stage conversion, pathogenesis, and tissue cyst formation in mice fed bradyzoites of different strains of Toxoplasma gondii. Journal of Eukaryotic Microbiology 44, 592602.[ISI][Medline]