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Antimicrobial Agents and Chemotherapy, December 2003, p. 3853-3858, Vol. 47, No. 12
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.12.3853-3858.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Activity of 9-(S)-[3-Hydroxy-2-(Phosphonomethoxy)Propyl]Adenine against Schistosomiasis mansoni in Mice

Sanaa Botros,1 Samia William,1 Olfat Hammam,1 Zdenek Zídek,2* and Antonín Holy3

Theodor Bilharz Research Institute, Giza, Egypt,1 Institute of Experimental Medicine,2 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of The Czech Republic, Prague, Czech Republic3

Received 3 June 2003/ Returned for modification 18 July 2003/ Accepted 19 September 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and METHODS
 Results
 Discussion
 References
 
The activity of the acyclic nucleotide analogue 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine [(S)-HPMPA] against Schistosoma mansoni was investigated in mice. The compound was injected intraperitoneally, usually on two or five consecutive days, at 10 to 20 mg/kg of body weight/day. The treatment started before, at the time of, and after the onset of egg laying (oviposition) by S. mansoni. The animals were killed from 7 to 40 days after the cessation of treatment. Significant reductions in the total numbers of female and coupled worms were found. Female fecundity and both hepatic and intestinal egg loads were suppressed. These effects were more pronounced with dosing regimens launched before the time of oviposition. The complete disappearance of immature eggs and a significant reduction to the complete absence of mature eggs, with 99 to 100% of the eggs being dead, were produced. No hepatic egg-induced granulomas were present in mice treated at the time of oviposition, and the granulomas were smaller in mice treated before S. mansoni oviposition. These preliminary findings extend the knowledge of the antiparasitic properties of (S)-HPMPA.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and METHODS
 Results
 Discussion
 References
 
The only antibilharzial drug which is effective against the four main schistosomes pathogenic for humans is praziquantel (PZQ) (9). It has minimal side effects (18), but the control of schistosomiasis with PZQ at the population level faces problems, including the development of drug resistance (23). Reduced cure rates and the failure of treatment after PZQ treatment have been reported in patients (8, 14, 22). The resistance of schistosomes has been demonstrated experimentally, in which it has been shown that worms exposed to subcurative doses of PZQ produce parasites less sensitive to the drug (7). When Schistosoma mansoni eggs from PZQ-resistant Egyptian villagers were used to establish infection-specific isolates in mice, the worms rather than the host factors were suggested to be responsible for the reduced sensitivity to PZQ (13, 34). In addition, PZQ does not prevent reinfection and sometimes even enhances the rate of reinfection, especially in young adults (2). Moreover, PZQ is not ovicidal, nor is it effective against young stages of the parasite (29). The development and use of new safe and effective drugs thus remain urgent challenges until a successful vaccine is produced.

Acyclic nucleotide analogs are a novel group of biologically active compounds developed primarily as virostatic compounds (12). Their major mechanism of action is the inhibition of virus-induced DNA polymerases and/or reverse transcriptases (20). Due to this mode of action, they have also been suggested to be prospective targets for antiparasitic therapy (30). Indeed, several acyclic nucleotide analogues modified at the heterocyclic base and/or side chain bearing the phosphonate group, namely, theS enantiomer of 9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine [(S)-HPMPA], the S enantiomer of 9-[3-hydroxy-2(phosphonomethoxy)propyl]2,6-diaminopurine, 9-[2-(phosphono methoxy)ethyl]adenine (PMEA), and 9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine, were found to possess antitrypanosomal activities (17). One of the most effective analogues proved to be (S)-HPMPA, which is effective against extracellular Trypanosoma brucei rhodesiense, T. b. gambiense, as well as multidrug-resistant T. b. brucei, Trypanosoma congolense, and Trypanosoma evansi (16). (S)-HPMPA and its derivative, (S)-3-deaza-HPMPA, also inhibit the growth of Plasmodium falciparum and Plasmodium berghei, while many other acyclic nucleotide analogues, including PMEA, are devoid of antiplasmodial activities (5, 30, 31).

(S)-HPMPA can thus be considered to exhibit unique antiparasitic activity. The aim of the preliminary study described here was to screen (S)-HPMPA for its potential activity against S. mansoni in a murine model of infection.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and METHODS
Results
 Discussion
 References
 
Animals. Male CD-1 Swiss albino mice (weight, 20 ± 2 g), bred and maintained under conventional conditions at the experimental animal research unit of the Schistosome Biological Supply Program at Theodor Bilharz Research Institute (Giza, Egypt), were used. They were fed a standard commercial pelleted diet. All animal experiments were conducted in accordance with valid international guidelines for animal experimentation.

Schistosomiasis induction. The livers and intestines of infected mice were digested and then filtered through sieves with different mesh size openings. Eggs were collected, and then dechlorinated water was added. The miracidia that hatched from the eggs were used to infect Biomphalaria alexandrina snails (size, 3 to 5 mm). The snails were infected en mass. The plates were left under ceiling illumination for 3 to 5 h at 25 to 27°C. At the end of the exposure time, the snails were collected and placed in plastic trays with a proper diet. Cercariae from at least 50 shedding B. alexandrina snails were used to infect the mice. Each mouse was infected with 100 S. mansoni cercariae by using a body immersion technique (Y. S. Liang, J. I. Bruce, and D. A. Boy, Proc. First Sino-Am. Symp., p. 34, 1987).

Drugs. PZQ (EMBAY 8440) was purchased from Bayer (Leverkusen, Germany) and E. Merck (Darmstadt, Germany). (S)-HPMPA was synthesized in-house (by A. Holy) by a previously described procedure (11). For the in vitro studies, the compounds were prepared as 10 mM stock solutions in dimethyl sulfoxide and distilled water, respectively. They were further diluted to the desired concentrations with distilled water. For the in vivo experiment, (S)-HPMPA was dissolved in sterile distilled water to give a stock solution of 1 mg/ml. It was freshly prepared before intraperitoneal administration.

Dosing scheme. Two major criteria were taken into account in planning the experimental design (Fig. 1): (i) the onset of (S)-HPMPA administration and (ii) the dose. The time of administration was used to take into account the basic schistosome life cycle stages, because the time of exposure of schistosomes to a drug may often be of greater importance than the dose applied (9). The mice were thus divided into three groups. The point of concern was the maturity of the parasites and, hence, the capacity of female worms to lay eggs (oviposition). Treatment was started on day 49 postinfection (i.e., after oviposition), day 30 (i.e., at the time of onset of oviposition), and day 25 (i.e., before oviposition). No data on the potential antischistosomal activity of (S)-HPMPA are available from pilot studies. The decision on the doses to be used was thus primarily guided by experiments describing the activities of (S)-HPMPA against other parasites (16). (S)-HPMPA was injected intraperitoneally at doses of 10 to 50 mg/kg of body weight/day (Fig. 1). The injections were given on two (group I) or five (groups III, IV, V, VI, and VII) consecutive days or every other day (eight injections; group II). The dose of 50 mg/kg was fractionated into four injections applied every 3 h on the same day (groups VIII and IX). The animals were killed at various times postinfection (49 to 72 days) and posttreatment (7 to 40 days).



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  		FIG. 1. Experimental design. The mice were infected with S. mansoni (100 cercariae per mouse) on day 0. (S)-HPMPA was injected intraperitoneally at doses of 10 ({triangledown}) and 20 ({blacktriangledown}) mg/kg/day. The injections were given on two (group I) or five (groups III, IV, V, VI, and VII) consecutive days or every other day (eight injections; group II). The dose of 50 mg/kg/day was fractionated into four injections given every 3 h on the same day (groups VIII and IX) ({diamond}). The HPMPA treatment was started on day 49 postinfection (after oviposition) (A), day 30 (at the time of oviposition) (B), or day 25 (before oviposition) (C). The animals were killed at the indicated time intervals, i.e., 49, 56, or 70 to 72 days postinfection (•). The controls (i.e., infected animals not treated with HPMPA; data not shown) were also killed at these time points.

 
After the mice were killed, the hepatic and portomesenteric vessels were perfused to study worm load and sex. Parts of the livers were collected and fixed in 10% formalin for histopathological examination and for measurement of granuloma diameters. The rest of the liver and intestinal fragment tissues were used for the recovery of eggs and determination of the percentage of eggs in each developmental stage.

Worm burden and distribution. Worm burden and sex were studied after perfusion of the hepatoportomesenteric vessels (6).

Egg developmental stages (oogram pattern). After perfusion, the small intestine was wholly separated and transferred to a petri dish. Three fragments (length, 1 cm each) of the small intestine were cut longitudinally, rinsed in saline, slightly dried on filter paper, and then placed between a slide and a coverslip. The preparations were examined under the low power of a microscope, and the stage of each egg in each fragment was recorded. Three fragments were obtained from each animal and examined, and the mean number of eggs in each developmental stage was calculated (27).

Tissue egg load. The number of eggs per gram of tissue was studied by weighing a piece of liver or small intestine, which was then digested and incubated overnight in 5% KOH. The hepatic and intestinal tissue egg loads were determined by multiplying the average number of eggs in each 1-ml sample by the total volume of KOH and then dividing that value by the weight of the sample to yield the number of eggs per gram of tissue (19).

Hepatic histopathology and granuloma measurement. For granuloma measurements, five sections (thickness, 5 µm each), each of which was 250 µm from the preceding one, were prepared and stained with Masson trichrome stain. An ocular micrometer was used to measure noncontiguous granulomas, each of which contained a single egg in its center. The mean diameter of each granuloma was obtained by measuring the diameter of the lesion twice, with the second measurement made at a right angle to the first one. The overall mean granuloma diameter represents the measurements for 150 to 210 lesions from five to seven animals per group. The cellular profile, the state of the S. mansoni eggs, and the associated histopathological changes were examined in three hematoxylin-eosin-stained sections.

The percentage of degenerated ova was calculated from the number of degenerated miracidia (acellular or partially or completely degenerated, leaving an empty shell) within the ova and the total number of granulomas per mouse by the following formula: (mean number of degenerated ova/mean number of granulomas) x 100.

Reversible cell injury (i.e., hydropic degeneration and necrosis) was studied by calculating the percentage of the cell area expressing either of these changes per section in five microscopic fields (magnification, x40). The mean value per mouse was obtained first, and then the mean for the group was obtained. The cells were examined for hydropic degeneration by looking for small clear vacuoles and/or punched-off areas in hepatocytes as a result of water accumulation. The cells were examined for necrosis by looking for a glassy cell appearance as a result of a loss of cell glycogen particles together with eosinophilia (3).

Statistical analysis. Analysis of variance, Bartlett's test for homogeneity of variances, a subsequent Dunnett's multiple-comparison test, and graphical presentation of the data were done by using the Prism program (GraphPad Software, San Diego, Calif.). Several control groups of animals (which were infected but not treated with the drug) were killed 49, 56, and 70 to 72 days postinfection. Data for the control animals were combined for comparison with the data for treated animals, since no significant differences in the parameters mentioned above were found among the animals in the control groups.


   RESULTS
Top
 Abstract
 Introduction
 Materials and METHODS
 Results
Discussion
 References
 
Total number of worms after (S)-HPMPA treatment. Among the control groups used in the study, there were no significant differences in the total number of unpaired male worms (overall mean, 15.8 ± 1.5; P = 0.45), unpaired female worms (overall mean, 13.5 ± 0.6; P = 0.23), and coupled worms (overall mean, 12.4 ± 0.6; P = 0.06). (S)-HPMPA treatment, irrespective of the dose or the onset or duration of treatment, led to significant reductions (with a few exceptions) in the total number of female and coupled worms (overall, 30 and 40%, respectively) but not in the total number of male worms (12%) (Fig. 2).



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  		FIG. 2. Effects of (S)-HPMPA on total number of S. mansoni organisms in infected mice. (S)-HPMPA was injected intraperitoneally at the indicated doses. Drug administration started after oviposition (day 49), at the time of oviposition (day 30), or before oviposition (day 25). The animals were killed at various times postinfection (56 to 72 days) and posttreatment (days 7 to 40). The bars and the lines above the bars indicate means ± standard errors of the means. The horizontal solid and dotted lines represent the mean and the 95% confidence limits, respectively, for the controls (i.e., infected animals not treated with HPMPA). * and **, statistically significant at P < 0.05 and P < 0.01, respectively.

 
Female fecundity and tissue egg load. The influence of (S)-HPMPA on the ability of S. mansoni females to lay eggs (fecundity) depended on the dosing regimen applied (Fig. 3). Overall, the average number of eggs per female in the infected untreated animals was 4,126 ± 430. The suppressive effect of (S)-HPMPA was statistically significant when the treatment started before the time of oviposition (i.e., 25 days postinfection). However, only the highest doses (i.e., 20 mg/kg/day given on five consecutive days and 50 mg/kg fractionated into four injections [12.5 mg/kg each] given every 3 h on the same day) reduced the fecundity. The effect persisted for at least 19 days posttreatment (Fig. 3, groups IV and VIII) but could not be detected at 40 days posttreatment (Fig. 3, groups V and IX). In principle, the same results were detected for hepatic and intestinal egg loads, although the intestinal egg load was more affected (Fig. 3).



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  		FIG. 3. Effects of (S)-HPMPA on female fecundity and egg loads in mouse tissue. The experimental conditions are as described in the legend to Fig. 2. The bars and the lines above the bars indicate means ± standard errors of the means. The horizontal solid and dotted lines represent the mean and the 95% confidence limits, respectively, for the controls (i.e., infected animals not treated with HPMPA). * and **, statistically significant at P < 0.05 and P < 0.01, respectively.

 
Quality of eggs and ovicidal activity of (S)-HPMPA. No immature eggs were present in mice treated with (S)-HPMPA, irrespective of the dosing regimen, when the mice were evaluated over 19 days posttreatment. At 40 days posttreatment, the immature eggs reappeared (groups V, VII, and IX), but the frequency was significantly lower compared to that in the controls (Fig. 4). Mature eggs were statistically significantly reduced in number or were completely missing in animals receiving (S)-HPMPA at 20 mg/kg/day for five consecutive days (group IV) or 50 mg/kg fractionated into four injections (12.5 mg/kg each) given every 3 h (group VIII) when treatment was started before oviposition (i.e., on day 25 postinfection). The oogram showed enormous increases in the proportion of dead eggs, which reached or closely approached 100%, although dead eggs were detected less frequently at day 40 posttreatment, when immature eggs reappeared (groups V, VII, and IX).



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  		FIG. 4. Effects of (S)-HPMPA on development of S. mansoni eggs. The experimental conditions are as described in the legend to Fig. 2. The bars and the lines above the bars indicate means ± standard errors of the means. The horizontal solid and dotted lines represent the mean and the 95% confidence limits, respectively, for the controls (i.e., infected animals not treated with HPMPA). * and **, statistically significant at P < 0.05 and P < 0.01, respectively.

 
Development of schistosomal hepatic granuloma. Granuloma formation was evaluated in animals given (S)-HPMPA on day 25 postinfection and killed 19 or 40 days posttreatment (Fig. 5). All dosing regimens led to significant decreases in the frequency of granulomas and/or to substantial reductions in granuloma diameters (on average, a 46% reduction in comparison with the size of the granuloma in untreated infected controls, which was a mean of 276.6 ± 4.4 µm). The granulomas were cellular, with predominant eosinophils and neutrophils. Most of the S. mansoni eggs from the treated animals showed signs of severe degeneration (85% versus 11.4% for the controls). The necrosis in the hepatocytes and granulomas was focal, and necrosis was found in 50% of (S)-HPMPA-treated mice, whereas it was found in 29% of the controls. There was no significant change in hydropic degeneration between control infected animals (57%) and (S)-HPMPA-treated ones (60 to 70%). The hepatic lobular architecture was found to be preserved in both control and (S)-HPMPA-treated mice.



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  		FIG. 5. Effects of (S)-HPMPA on hepatic schistosomal granuloma. The experimental conditions are as described in the legend to Fig. 2. The drug was applied on day 25 postinfection. The bars and the lines above and below the bars indicate means ± standard errors of the means. The horizontal solid and dotted lines represent the mean and the 95% confidence limits, respectively, for the controls (i.e., infected animals not treated with HPMPA). a, typical granulomas were found in only five of seven animals; b, typical granulomas were found in only two of seven animals; **, statistically significant at P < 0.01, respectively.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and METHODS
 Results
 Discussion
References
 
The acyclic adenosine analogue (S)-HPMPA was originally developed as an antiviral agent (4, 33), but it also proved to possess antitrypanosomal (16, 17) and antiplasmodial (5, 30, 31) activities. We have found that it also interferes with the basic life cycles of S. mansoni in the mouse model. Various parasitological criteria indicate the in vivo antischistosomal effects of certain (S)-HPMPA dosing regimens: it caused significant reductions in worm loads, tissue egg loads, and the frequency of egg developmental stages. Most prominently, (S)-HPMPA treatment resulted in the nearly complete disappearance of mature and immature eggs. The most effective times of the start of treatment were 25 and 30 days postinfection, i.e., before and at the time of oviposition by female worms, although delayed (S)-HPMPA administration (at 49 days postinfection, i.e., after oviposition) still remained considerably effective.

The main targets for the action of (S)-HPMPA are the female worms in terms of either the load of female worms or their ability to lay eggs. In contrast to the effects of the recognized antischistosomal drug PZQ, which kills adult schistosomes (29), the killing effect of (S)-HPMPA is weaker. In this respect, female S. mansoni worms seemed to be more sensitive than male worms. The decline in the number of females was found even in the group of animals given the lowest total dose, i.e., two daily injections of 10 mg of (S)-HPMPA per kg. Dramatic alterations in the oogram patterns were produced, although they were less pronounced in mice killed at 40 days posttreatment; the residual worms recovered and again started to lay eggs. Thus, (S)-HPMPA transiently affects the fecundity of the worms and/or the viability of the eggs. It remains to be determined whether this transient effect could be overcome by more appropriate dosing, e.g., by repeated administration of (S)-HPMPA at 2-week intervals.

In schistosomiasis, intact eggs, whether they are living or dead, can induce granulomatous reactions, but shells or miracidia cannot (1). As a rule, the livers of (S)-HPMPA-treated mice showed no schistosomal granulomas or showed greatly reduced granuloma diameters and a preserved typical lobular architecture. Apparently, residual eggs (i.e., eggs trapped in the intravascular space) and eggs that are "silent" in tissue (i.e., eggs that are incapable of evoking a tissue reaction) were affected in situ by (S)-HPMPA and lost the capability to induce granulomatous responses in liver tissue. This could result from metabolic impairment of the embryonated eggs and/or from direct killing of the eggs, which probably leads to defects in the secretion of soluble egg antigen.

Granuloma formation in schistosomiasis is a manifestation of delayed-type hypersensitivity to an antigenic material released by S. mansoni eggs (SEA)(25) that peaks at 8 weeks postinfection. It is associated with increased T-cell proliferation and the production of inflammatory cytokines (32). We have found that eosinophils are the preponderant cell type in the greatly reduced liver granulomas of (S)-HPMPA-treated mice. These cells are known to play a major role in ovum destruction both in vitro (15) and in vivo (26). Amelioration of hepatic pathology and the presence of small granulomas with predominant eosinophils as a result of accelerated egg destruction upon repeated administration of SEA have been reported (10). Therefore, the possibility that the granuloma hyporesponsiveness observed after (S)-HPMPA treatment could at least partially depend on immune interventions cannot be ruled out. Interestingly, a number of acyclic nucleoside phosphonates possess immunomodulatory properties (35).

It has been suggested that after (S)-HPMPA is diphosphorylated by cellular enzymes, it acts as an antiviral by competitively inhibiting the DNA polymerase- and reverse transcriptase-catalyzed incorporation of natural triphosphate nucleotides into DNA (12, 24). Others have suggested that related acyclic nucleotide analogues act as alternative substrates, leading to DNA chain termination or reduced levels of DNA chain elongation (21). It was shown that diphosphorylated (S)-HPMPA inhibits DNA polymerase and the growth of Plasmodium spp. Even though many other related purine- or pyrimidine base-modified analogues of the 9-(S)-3-hydroxy-2-(phosphonomethoxy)propyl series are inhibitors of DNA polymerases as well, they do not inhibit parasite growth (5, 30). This means that (S)-HPMPA is the only analogue which enters the cell, is the only one which is activated by phosphorylation, or is the only inhibitor of parasite DNA polymerases among the many acyclic nucleotide analogues that have been investigated. It is difficult to accept one of these explanations. It is probable that still another mode of action applies solely to parasites and remains to be uncovered. One such possibility could be purine nucleoside phosphorylase. More than a decade ago it was demonstrated that (S)-HPMPA and its mono- and diphosphate forms are strong inhibitors of purine nucleotide phosphorylase and severely disturb the purine cell pool (28). This enzyme is often altered in parasites, and it is essential for the purine metabolic cycle. In our opinion, either of these alternatives is worthy of further research to elucidate the underlying mechanism(s) of the antischistosomal activity of (S)-HPMPA.

In conclusion, the activity of (S)-HPMPA against schistosomes was recorded over a reasonably wide range of the life cycle. The drug mainly affects the schistosome eggs. The underlying mechanism(s) of the effects remains to be firmly elucidated. Presumably, it may be due to a direct assault on female worms, thus diminishing their numbers or their ability to lay eggs, although a direct ovicidal action cannot be excluded.


   ACKNOWLEDGMENTS
 
The work was supported by grant 22 from the Academy of Scientific Research of Egypt, grants 305/00/0048 and 305/03/1470 from the Grant Agency of the Czech Republic, and the program of targeted projects of the Academy of Sciences of the Czech Republic (grant S4055109). This work was performed as a part of research projects of the Theodor Bilharz Research Institute, the Institute of Organic Chemistry and Biochemistry (project 4055905), and the Institute of Experimental Medicine (project AVOZ5008914).


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Experimental Medicine, Academy of Sciences, Vídenská 1083, 142 20 Prague 4, Czech Republic. Phone: (420) 241 062 720. Fax: (420) 241 062 109. E-mail: zidekz{at}biomed.cas.cz. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and METHODS
 Results
 Discussion
 References
 

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Antimicrobial Agents and Chemotherapy, December 2003, p. 3853-3858, Vol. 47, No. 12
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.12.3853-3858.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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