Ketolide ABT-773 is active against Toxoplasma gondii

Anis A. Khana,b, Fausto G. Araujoa, J. Carl Craftc and Jack S. Remingtona,b,*

a Research Institute, Palo Alto Medical Foundation, Ames Building, 795 El Camino Real, Palo Alto, CA; b Stanford University School of Medicine, Palo Alto, CA 94301; c Abbott Laboratories, Chicago, IL, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ABT-773 is active in vitro and in vivo against Toxoplasma gondii. It inhibited replication of RH strain tachyzoites in human foreskin fibroblasts. Mice infected intraperitoneally with tachyzoites and treated orally with 25, 50 or 100 mg/kg/day of ABT-773 for 10 days had 20% (P = 0.016), 50% (P = 0.003) and 100% (P = 0.001) survival, respectively. Remarkable and highly significant survival was also noted in mice infected orally with strain C56 cysts and treated with ABT-773. Thus, ABT-773 may be useful for therapy of human toxoplasmosis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Toxoplasmosis remains a significant problem among organ transplant recipients, patients with AIDS who have pre-existing latent infection with toxoplasma and the fetuses of women who acquire Toxoplasma gondii infection during gestation. Although treatment of the acute infection in AIDS patients and organ transplant recipients with pyrimethamine and either sulfadiazine or clindamycin has been successful in most cases, the relatively high incidence of toxicity associated with these drug combinations has frequently resulted in a reduction in the dosage or discontinuation of one or both drugs in the combination.1 Therefore, there is a need to find new drugs with increased potency and lower toxicity to treat all forms of toxoplasmosis. ABT-773 [11-amino-11-deoxy-3-oxo-5-O-desosaminyl-6-O-(1'-(3'-quinolyl-2'-propenyl)] erythronolide A 11,12-cyclic carbamate) is a novel ketolide antimicrobial agent. Since macrolides, azalides, lincosamides, streptogramins and ketolides have activity against T. gondii,25 we investigated the activity of ABT-773 against T. gondii both in vitro in cultured cells and in vivo in murine models of the infection.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
T. gondii

Tachyzoites of the RH strain were obtained from the peritoneal cavities of mice that had been infected 2 days earlier; cysts of the C56 strain were from the brains of chronically infected mice.6

Mice

Swiss Webster female mice, 5–6 weeks old and weighing 18–20 g at the beginning of each experiment, were purchased from Taconic Laboratories, Germantown, NY, USA.

Drug

ABT-773 (Abbott Laboratories, Abbott Park, IL, USA) was dissolved in sterile double-distilled water or 0.25% carboxymethyl cellulose for the in vitro and in vivo experiments, respectively.

In vitro experiments

The effect of ABT-773 on T. gondii was defined as the capacity of the drug to inhibit intracellular replication of T. gondii and was determined using the [3H]uracil incorporation technique.6 Human foreskin fibroblasts (HFF) (ATCC HS 68) were grown to confluence in 96-well, flat-bottomed microtitre plates and infected with 6 x 104 RH tachyzoites. Four hours after infection, the monolayers were washed and various concentrations of ABT-773 (0.00–25.00 mg/L) were added. Addition of the drug marked the beginning time point. Four hours before harvesting the cells, [5,6-3H]uracil (1 µCi/well) was added. Cells were harvested at 48 h and the total amount of radioactivity incorporated was determined with a scintillation counter.

The cytotoxicity of ABT-773 for HFF was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide cell proliferation assay (MTT assay, Cell Titer 96 Kit; Promega Corp., Madison, WI, USA).

In vivo experiments

Mice were infected intraperitoneally with 2.5 x 103 tachyzoites of the RH strain of T. gondii. Treatment with increasing single daily doses of oral ABT-773 was initiated 24 h after infection and was continued for 10 days. Control mice were infected and treated with the diluent alone. Mice were observed twice daily (for 30 days) for morbidity (ruffled fur and loss of weight) and mortality. There were five mice in the control group and 10 mice in each treatment group. For the oral infection model of toxoplasmosis, mice were administered 10 cysts of the C56 strain per os and treated with increasing single daily doses of oral ABT-773 starting 3 days after infection and continuing for 10 days. An additional lower dose of ABT-773 (12.5 mg/kg) was used because previous work with ketolides indicated that lower doses were effective in mice infected by the oral route.4 All other experimental details were same as in the RH experiment.

Statistical analysis

P values were obtained by the log-rank test of the Kaplan– Meier product-limited survival analysis (StatView; Abacus Concepts, Berkeley, CA, USA) and were considered statistically significant at P <= 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ABT-773 inhibited intracellular replication of T. gondii in a concentration-dependent manner from 0 to 25 mg/L (Figure 1aGo). The IC50 was 1.99 mg/L (correlation coefficient = 0.982) following 48 h exposure. ABT-773 had no effect on growth of host cells at concentrations that were highly active against T. gondii (Figure 1bGo).



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Figure 1. Effect of ABT-773 on (a) intracellular replication of RH strain of T. gondii in vitro determined by [3H]uracil incorporation and (b) mitochondrial activity of host HFF cells in vitro determined by the MTT cellular proliferation assay as measured by absorbance at 570 nm. Results are presented as means + S.D. from triplicate assays.

 
Oral treatment of mice infected intraperitoneally with RH tachyzoites resulted in a significant dose-dependent protection against death (Figure 2aGo). Doses of 25, 50 or 100 mg/kg/day protected 20% (P = 0.016), 50% (P = 0.003) or 100% (P = 0.001) of infected mice, respectively. All control mice died by day 10 of infection. Oral treatment of mice infected orally with C56 cysts also resulted in significant protection against death (Figure 2bGo). Doses of 12.5, 25, 50 or 100 mg/kg/day protected 40% (P = 0.019), 90% (P < 0.0001), 80% (P < 0.0001) or 90% (P < 0.0001) of infected mice, respectively. All control mice died by day 17 of infection. Mice treated with the highest dose did not show any significant signs of morbidity. Cysts were observed in the brains of the surviving mice at the end of the 30 day period.



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Figure 2. Survival in mice infected (a) intraperitoneally with tachyzoites of the RH strain or (b) orally with cysts of the C56 strain and treated with ABT-773. Once-daily treatment was initiated 1 (a) or 3 days (b) after infection and was continued for 10 days. There were five mice in the control group and 10 in each of the treatment groups. {blacksquare}, Control; {triangleup} (panel b only), 12.5 mg/L; {blacktriangledown}, 25 mg/kg; {circ}, 50 mg/kg; •, 100 mg/kg.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results described above reveal that ABT-773 has significant activity against T. gondii both in vitro and in two murine models of acute toxoplasmosis. In vivo, ABT-773 was active against two different strains of T. gondii and was active against infections in mice resulting from two different routes of infection. Although treatment was protective against death, it did not prevent formation of cysts in the brain of mice infected with C56 cysts. In addition, although there was a clear dose-dependent protection following infection with the RH strain, such an effect was not clearly observed following infection with the C56 strain. One of the reasons for this difference could be the ability to control the inoculum size precisely with tachyzoites but not with cysts. Variation in inoculum size is introduced because of cyst size, imprecise counting and the variable numbers of organisms in each cyst.

Ketolides are an advanced generation of macrolide antibiotics that differ from erythromycin A in that they harbour a 3-keto group instead of an l-cladinose group and are more acid-stable than clarithromycin, roxithromycin or azithromycin.7 It has been previously reported that macrolides, azalides and lincosamides have activity against T. gondii.2,3 More recently, we reported that the ketolides4 and the streptogramins,5 new members of the macrolide family, have activity against T. gondii. Whereas only high doses of the earlier macrolides and azalides had significant activity against T. gondii in vivo (azithromycin, 200 mg/ kg/day; roxithromycin, 500 mg/kg/day; clarithromycin, 300 mg/kg/day; and clindamycin, 300 mg/kg/day),2,3 the advanced generation ketolides (HMR 3647, 30 mg/ kg/day; HMR 3004, 40 mg/kg/day) and streptogramins (quinupristin–dalfopristin; 100 mg/kg/day) are much more potent.4,5 The potency of ABT-773 for T. gondii is comparable to that of the earlier ketolides and quinupristin– dalfopristin against T. gondii. The optimal dose in mice appears to be 100 mg/kg. This dose was most effective against infection with either tachyzoites or cysts. Since oral infection with cysts is the natural route of infection in humans and ABT-773 at 25 mg/kg was highly effective in mice; allometrically, an equivalent dose in a 60 kg human would be 125 mg. However, whether this dose would be effective against toxoplasmosis in humans will depend on pharmacokinetic and pharmacodynamic factors and must be clinically determined.

Widespread use of macrolides has given rise to a rapid increase in the rates of resistance of clinical isolates of Streptococcus pneumoniae and other bacterial pathogens to these antibiotics. ABT-773, a novel ketolide with a quinolylallyl moiety in combination with a keto group at the 3-position and a carbamate group at the 11,12-position, has more potent antibacterial activity than other macrolides against Gram-positive bacteria in vitro and has activity against resistant respiratory pathogens.8 The mechanism of action of ABT-773 in bacteria is by interaction with 23S ribosomal RNA and subsequent concentration-dependent inhibition of protein synthesis.9 The mechanism of action of ABT-773 against T. gondii is not known. However, it has been suggested that ribosomes encoded by the plastid-like ribosomal genes found in T. gondii may be sensitive to the lincosamide/macrolide class of antibiotics, and may serve as a functional target for protein synthesis inhibitors in T. gondii and related parasites.10

Our results suggest that ABT-773, with its potent in vivo activity against T. gondii, may be useful for therapy of toxoplasmosis in humans.


    Acknowledgments
 
This work was supported by a US Public Health Services grant AI04717, NIH contract N01-AI-35174, and grants from Abbott Laboratories.


    Notes
 
* Corresponding author. Tel: +1-650-853-6061; Fax: +1-650-329-9853. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Liesenfeld, O., Wong, S. Y. & Remington, J. S. (1999). Toxoplasmosis in the setting of AIDS. In Textbook of AIDS Medicine, 2nd edn, (Merigan, T. C., Bartlett, J. G. & Bolognesi, D., Eds), pp. 225–59. Lippincott Williams & Wilkins, Baltimore, MD.

2 . Araujo, F. G. & Remington, J. S. (1992). Recent advances in the search for new drugs for treatment of toxoplasmosis. International Journal of Antimicrobial Agents 1, 153–64.

3 . Araujo, F. G., Prokocimer, P., Lin, T. & Remington, J. S. (1992). Activity of clarithromycin alone or in combination with other drugs for treatment of murine toxoplasmosis. Antimicrobial Agents and Chemotherapy 36, 2454–7.[Abstract]

4 . Araujo, F. G., Khan, A. A., Slifer, T. L., Bryskier, A. & Remington, J. S. (1997). The ketolide antibiotics HMR 3647 and HMR 3004 are active against Toxoplasma gondii in vitro and in murine models of infection. Antimicrobial Agents and Chemotherapy 41, 2137–40.[Abstract]

5 . Khan, A. A., Slifer, T. R., Araujo, F. G. & Remington, J. S. (1999). Quinupristin–dalfopristin is active against Toxoplasma gondii. Antimicrobial Agents and Chemotherapy 43, 2043–45.[Abstract/Free Full Text]

6 . Araujo, F. G., Huskinson, J. & Remington, J. S. (1991). Remarkable in vitro and in vivo activities of the hydroxynaphthoquinone 566C80 against tachyzoites and tissue cysts of Toxoplasma gondii. Antimicrobial Agents and Chemotherapy 35, 293–9.[ISI][Medline]

7 . Bryskier, A., Agouridas, C. & Chantot, J. F. (1997). Ketolides: new semisynthetic 14-membered-ring macrolides. In Expanding indications for the new macrolides, azalides, and streptogramins, (Zinner, S. H., Young, L. S., Acar, J. F. & Neu, H. C., Eds), pp. 39–50. Marcel Dekker, Basel.

8 . Ma, Z., Clark, R. F. & Or, Y. (1999). Design, synthesis and characterization of ABT-773: a novel ketolide highly active against multidrug resistant pathogens. In Program and Abstracts of the Thirty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 2133, p. 345. American Society for Microbiology, Washington, DC.

9 . Cao, Z., Hammond, R., Pratt, S., Saiki, A., Lerner, C. & Zhong, P. (1999). Mechanism of action for novel ketolide ABT773. In Program and Abstracts of the Thirty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 2135, p. 346. American Society for Microbiology, Washington, DC.

10 . Beckers, C. J. M., Roos, D. S., Donald, R. G. K., Luft, B. J., Schwab, J. C., Cao, Y. et al. (1995). Inhibition of cytoplasmic and organellar protein synthesis in Toxoplasma gondii. Implications for the target of macrolide antibiotics. Journal of Clinical Investigation 95, 367–76.[ISI][Medline]

Received 22 December 1999; returned 15 March 2000; revised 6 April 2000; accepted 2 May 2000