Evaluation of two inhibitors of invasion: LY311727 [3-(3-acetamide-1-benzyl-2-ethyl-indolyl-5-oxy)propane phosphonic acid] and AEBSF [4-(2-aminoethyl)-benzenesulphonyl fluoride] in acute murine toxoplasmosis

Rafael Buitrago-Rey1,2, Jorge Olarte2 and Jorge Enrique Gomez-Marin1,3,*

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.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We examined the effect of an inhibitor of secretory phospholipase A2 type II (LY311727) and of a specific inhibitor of serine proteases (AEBSF) in a murine model of acute toxoplasmosis. LY311727 did not afford any significant protection and produced earlier mortality compared with untreated mice at a dose of 100 mg/kg. In contrast, AEBSF demonstrated a significant increase in length of survival, and this effect was enhanced when AEBSF was administered with non-protective doses of pyrimethamine.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Toxoplasma gondii is an intracellular obligate protozoan parasite that can infect almost all types of cell.1 Cell invasion is a critical step during infection of susceptible hosts by T. gondii. The parasite must gain entry into nucleated host cells for survival and replication. Although invasion is a logical target for antiprotozoal drugs, few antimicrobials have been developed to block this step.2 It has been demonstrated that phospholipase A2 (PLA2) enzymes3 and serine proteases4 play an important role in invasion by T. gondii.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Mice

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 16–17 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 Kaplan–Meier product limit method and P values were compared by the log-rank proportional Cox’s method.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
All untreated or vehicle-treated mice died between 6 and 8 days after infection. Pyrimethamine began to confer 100% survival at doses >35 mg/kg. Treatment with lower doses of pyrimethamine gave a mortality rate similar to that for controls. In three separate experiments all untreated and LY311727-treated mice died before day 9 (Figure 1). Moreover, percentage mortality was significantly higher at day 6 in mice treated with a dose of 100 mg/kg compared with untreated mice (P = 0.05), indicating a toxic effect of the drug at this dose.



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Figure 1. Percentage survival with (a) LY311727 alone at 0.2 (square), 2 (triangle), 5 (cross) or 15 (asterisk) mg/kg (diamond, control), or (b) LY311727 alone at 35 (circle), 61.5 (plus) or 100 (square) mg/kg (diamond, control), for treatment of mice with acute toxoplasmosis.

 
All untreated mice died by day 7 post-infection. AEBSF at doses of 38.4 (P = 0.04) or 76.8 mg/kg (P = 0.001) prolonged time to death in a statistically significant manner (Figure 2). When AEBSF 76.8 mg/kg was combined with a non-effective dose of pyrimethamine (10 mg/kg), the length of survival was increased in a statistically significant manner, compared with the same dose of AEBSF used alone (P = 0.00092). This suggests a synergic protective effect against Toxoplasma infection when the drugs were combined.



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Figure 2. Percentage survival with (a) AEBSF alone at 2.4 (square), 4.8 (triangle), 9.6 (plus) or 19.2 (asterisk) mg/kg (diamond, control), or (b) AEBSF 19.2 (circle) twice daily, 38.4 (circle) or 76.8 (minus) mg/kg, or pyrimethamine 10 mg/kg (open diamond) or a combination of AEBSF 76.8 mg/kg with pyrimethamine 10 mg/kg (plus) for treatment of mice with acute toxoplasmosis (filled diamond, control).

 
We used the first structure-based selective inhibitor of sPLA2 type II, which has no effect on cPLA2 activity.5,8 We tested this inhibitor, LY311727, in vivo, in order to detect a possible protective effect against toxoplasmosis, but no dose delayed mortality significantly; in fact, this agent led to an earlier mortality compared with untreated mice when it was used at a high dose (100 mg/kg). We would need to purify Toxoplasma sPLA2 in order to develop a specific and efficient inhibitor.

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.


    Acknowledgements
 
We thank Dr Michelich from Lilly Laboratories for providing us with LY311727, and Dr Jesús Cortes (veterinarian) for advice in the experiments with mice. This study was financed by funds from the ‘Instituto de Salud en el Tropico’, Universidad Nacional de Colombia.


    Footnotes
 
* Corresponding author. Tel/Fax: +57-674-60168; E-mail: jegomezmarin{at}hotmail.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Bonhomme, A., Bouchot, A., Pezzella, N., Gómez-Marin, J. E., Le Moal, H. & Pinon, J. M. (1999). Signalling during the invasion of host cells by Toxoplasma gondii. FEMS Microbiology Reviews 23, 551–61.[ISI][Medline]

2 . Katlama, C. (1996). Diagnosis and treatment of toxoplasmosis of the CNS in patients with AIDS. CNS Drugs 5, 331–47.[ISI]

3 . Gomez-Marin, J. E., Bonhomme, A., Guenounou, M. & Pinon, J. M. (1996). Role of interferon-{gamma} against invasion by Toxoplasma gondii in a human monocytic cell line (THP1): Involvement of the parasite’s secretory phospholipase A2. Cellular Immunology 169, 218–25.[ISI][Medline]

4 . Conseil, V., Soete M. & Dubremetz, J. F. (1999). Serine protease inhibitors block invasion of cells by Toxoplasma gondii. Antimicrobial Agents and Chemotherapy 43, 1358–61.[Abstract/Free Full Text]

5 . Schevitz, R. W., Bach, N. J., Carlson, D. G., Chirgadze, N. Y., Clawson, D. K., Dillard, R. D. et al. (1995). Structure-based design of the first potent and selective inhibitor of human non-pancreatic secretory phospholipase A2. Nature Structural Biology 2, 458–65. [ISI][Medline]

6 . Coombs, G. H. & Mottran, J. C. (1997). Parasite proteinases and amino acid metabolism: possibilities for chemotherapeutic exploitation. Parasitology 114, S61–80.[ISI][Medline]

7 . Megyeri, P., Nemeth, L., Pabst, K. M., Deli, M. A. & Abraham, C. S. (1999). 4-(2-Aminoethyl)benzenesulfonyl fluoride attenuates tumor-necrosis-factor-{alpha}-induced blood–brain barrier opening. European Journal of Pharmacology 374, 207–11.[ISI][Medline]

8 . Gomez-Marin, J. E., Bonhomme A. & Pinon, J. M. (1998). Une famille d’enzymes présentes des protozoaires aux mammiferes: comment les phospholipases A2 participent au processus d‘invasion de Toxoplasma gondii. L’Année Biologique 78, 185–202.

9 . Choi, W. Y., Nam, H. W. & Youn, J. H. (1989). Characterization of proteases of Toxoplasma gondii. Korean Journal of Parasitology 27, 161–73.

10 . Schwartzmann, J. D. & Saffer, L. D. (1992). How Toxoplasma gets in and out of host cells. In Subcellular Biochemistry Vol. 18: Intracellular Parasites, (Avila, J. L. & Harris, J. R., Eds), pp. 333–64. Plenum Press, New York, NY.





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