1Facultad de Medicina; 2Posgrado de Farmacología, Facultad de Ciencias, Universidad Nacional de Colombia; 3Centro de Investigaciones Biomedicas, Facultad de Ciencias de la Salud, Universidad del Quindio, Armenia (Q), Colombia
Received 23 July 2001; returned 18 November 2001; revised 18 December 2001; accepted 23 January 2002.
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Abstract |
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Introduction |
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The compound 3-(3-acetamide-1-benzyl-2-ethyl-indolyl-5-oxy)propane phosphonic acid (LY311727) is an inhibitor of secretory PLA2 (sPLA2) type II.5 Incubation with this compound before Toxoplasma infection in vitro reduced the invasion index by 50% when parasites were pre-treated.1 These effects were related to invasion and not to inhibition of intracellular growth as the reduction in infected cells was observed before the first division of the parasite.1 On the other hand, it was found that 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF), a specific inhibitor of serine proteases, prevented invasion of host cells by Toxoplasma.4 Serine proteinases that participate in invasion are found in other protozoa.6
Using drugs that have only an effect against invasion can leave intracellular parasites unaffected. However, a com-bination of drugs that act at different steps of parasitism (i.e. invasion and intracellular growth) might offer improvements in therapeutic efficacy against protozoa. Thus, in order to test the effect of these compounds in in vivo systems, we first examined in an experimental model of acute murine toxoplasmosis whether the addition of LY311727 or AEBSF would be beneficial in reducing mortality. Then, the com-pound showing a protective effect was used in combination with non-effective doses of pyrimethamine (which would have an effect on intracellular growth) in order to determine whether there would be an improved therapeutic effect. To our knowledge LY311727 and AEBSF are the only compounds that have been shown to act on the entry of Toxoplasma into host cells.
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Materials and methods |
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All experiments were approved by the Ethics Committee of the Animals Laboratory (Bioterio) at the Veterinary Faculty, Universidad Nacional de Colombia. Swiss ICR mice (Bio-terio, Universidad Nacional de Colombia, Bogota, Colombia) weighing 1617 g at the beginning of each experiment were used. Mice were housed 10 to a cage and given drinking water ad libitum.
T. gondii
Tachyzoites of the virulent RH strain maintained through serial intraperitoneal (ip) passages were used. For experimental infections, tachyzoites were harvested from mouse peritoneal fluids 72 h post-infection and purified by filtering on 3 µm diameter polycarbonate filters. The parasites were counted in a haemocytometer and their numbers were adjusted to 2 x 106/mL with saline and aliquots of 2.5 x 103 parasites were inoculated ip into each mouse.
Drugs
Pyrimethamine (lot: 9806000013; Laboratorios Roche S.A., Bogota, Colombia) was disolved in a solution of 0.25% carboxymethylcellulose and was administered orally with a feeding needle in a single daily dose of 70, 35, 10, 5, 2.5 or 1.25 mg/kg. LY311727 was kindly provided by E. Mihelich (lot: 34ANB; MO4-SER, Lilly Research Laboratories, Indianapolis, IN, USA) and was dissolved in a vehicle containing 5% dimethyl sulphoxide (DMSO), 5% ethanol and 30% polyethylene glycol-300 in water and was administered ip in a single daily dose of 0.2, 2, 5, 15, 35, 61.5 or 100 mg/kg. AEBSF (lot: 1879C; Interchim, Paris, France) was dissolved in sterile 0.89% saline solution and was administered ip in a single daily dose of 2.4, 4.8, 9.6, 19.2, 38.4 or 76.8 mg/kg. In addition, AEBSF at a dose of 19.2 mg/kg was tested using twice daily administration. These doses were selected on the basis of previous in vivo and in vitro reports.4,7
Experimental design
Mice injected with 2.5 x 103 parasites were randomly assigned to one of the treatment groups according to the treatment given: without drugs (control group), vehicle alone (vehicle control group), pyrimethamine alone at different doses, LY311727 alone at different doses, AEBSF alone at different doses or AEBSF 76.8 mg/kg plus pyrimethamine 10 mg/kg. Each treatment group consisted of 10 animals. Treatment was initiated 24 h after parasite inoculation and was continued for seven consecutive days. Mouse survival was monitored daily and continued in live mice until 15 days post-infection. All experiments were performed three times and the data shown represent the cumulative results.
Statistical analysis
The rates of survival in particular treatment groups were estimated by the KaplanMeier product limit method and P values were compared by the log-rank proportional Coxs method.
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Results and discussion |
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In contrast with the sPLA2 inhibitor, AEBSF treatment resulted in prolongation of the survival of mice that had been given a lethal T. gondii infection. There is little information available about Toxoplasma proteases.9,10 Recently it was shown that serine protease inhibitors (specifically 3, 4-dichloroisocoumarin and AEBSF) prevented invasion of host cells by Toxoplasma in vitro.4 AEBSF in a recent in vivo study was shown to be non-toxic and did not change vital parameters after its administration in newborn pigs.7 Serine protease contains a hydroxyl group in its catalytic site that initially attacks the carbonyl group of a peptide bond to form a tetrahedral intermediate before hydrolysis. In other parasites there are serine proteinases that seem to be important in their invasiveness. The opportunist parasite Acanthamoeba, for example, has been reported to produce a plasminogen activator. Serine proteases have also been implicated in the invas-ive mechanisms of Coccidian parasites.6 One link between serine proteinases and adhesiveness is that serine proteases activate integrin proteins.6 The importance of integrins in the adhesiveness of Toxoplasma has already been shown. Toxoplasma serine protease could be the parasite signal enabling binding of laminin to host-cell integrins.
In conclusion, this is the first report on the in vivo activity of drugs that specifically inhibit the invasion step of Toxoplasma. AEBSF treatment resulted in significantly prolonged time to death, and this effect was enhanced when AEBSF was used in combination with non-protective doses of pyrimethamine. However, AEBSF-treated mice died 12 days post-infection. Thus, further investigation of serine proteases in order to find compounds with a better activity is warranted.
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Acknowledgements |
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Footnotes |
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References |
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