1 Freie Universität Berlin, Institut für Pharmazie, Pharmazeutische Technologie, Biopharmazie und Biotechnologie, Kelchstraße 31, 12169 Berlin; 5 Georg August-Universität Göttingen, Institut für Organische Chemie, Tammannstraße 2, 35077 Göttingen; 6 Robert Koch-Institut, Abteilung für Infektionskrankheiten, Nordufer 20, 13353 Berlin, Germany; 2 National Animal Disease Center, Agricultural Research Services, United States Department of Agriculture, Ames, IA 50010; 3 Kansas State University, Biology Department, Ackert Hall, Manhattan, KS 66506-4901; 4 Wadsworth Center, Division of Infectious Diseases, David Axelrod Institute for Public Health, Albany, NY 12201-2002, USA
Received 5 November 2001; returned 22 March 2002; revised 17 May 2002; accepted 8 August 2002
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Abstract |
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Keywords: naphthoquinones, benzindazole-4,9-quinones, Cryptosporidium, in vitro, in vivo, antiprotozoal, cytotoxicity, drug testing
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Introduction |
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Although hundreds of drugs have been tested against cryptosporidiosis,5 none have proved effective enough to warrant extended clinical trials in AIDS patients. Drugs now used clinically include paromomycin,6 nitazoxanide,7 azithromycin plus paromomycin,8 roxithromycin9 and highly active antiretroviral therapy (HAART). Again, none is sufficiently efficacious to be recommended universally, and most of them either have significant side effects or do not prevent relapse.10 Therefore, there is an urgent need for both innovative and pharmaceutically improved drugs to treat this AIDS-associated disease.
A series of benzindazole-4,9-quinones (BIQs) initially developed to treat visceral leishmaniasis have recently been tested against C. parvum in vitro and in vivo. Structurally, BIQs are naphthoquinones that possess an additional imidazole ring.11 Naphthoquinones and other related quinoids are one of the major drug classes with significant activity against parasitic protozoa like Leishmania, Trypanosoma and Plasmodium.12 Many naphthoquinones have been isolated from plant or microbial sources, but in most cases their potential usefulness has been limited by cytotoxicity and low bioavailability. In contrast, the BIQs tested here showed no, or only moderate, cytotoxicity. For this reason, BIQs have been tested as lead compounds in a murine model of chronic cryptosporidiosis.
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Materials and methods |
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BIQs were synthesized by one of us (H. L.). Structures and >95% purity were determined by both nuclear magnetic resonance spectroscopy (H/C-NMR) and high-performance liquid chromatography (HPLC11,13). All compounds were first dissolved in dimethylsulphoxide (DMSO), aliquoted and then stored frozen until used, when they were diluted with phosphate-buffered saline (PBS) to the desired concentration (25100 µM).
In vitro testing for anticryptosporidial activity
A well-established in vitro assay was used to test the efficacy of these inhibitors against C. parvum.14 Briefly, human ileocaecal epithelial cells (HCT-8; ATCC CCL 244) were cultured in 75 cm2 tissue culture flasks in a maintenance medium consisting of RPMI 1640 supplemented with 10% Opti-MEM (Gibco-BRL), 2% fetal bovine serum (FBS) and 2 mM L-glutamine at 37°C in a humidified, 5% CO2-enriched atmosphere.1517 Ninety-six-well flat-bottomed microtitre plates were seeded with 5.0 x 104 HCT-8 cells/well and incubated for 1424 h. For infection, the maintenance medium was replaced by 100 µL/well parasite growth medium17 containing 3.0 x 104 sterilized oocysts. Non-viable, negative controls consisted of the same number of oocysts that had been frozen and thawed using liquid nitrogen and a 37°C water bath. Parasites were allowed to invade host cells for 90 min at 37°C, and any non-invading parasites were removed by rinsing once with warm PBS. After rinsing, 150 µL of fresh growth medium containing drugs at appropriate concentrations was added to each well. Negative controls contained drug diluent and growth medium only. Four to eight replicate wells were used for each experimental condition. All compounds were tested at least twice in separate experiments.
Infected HTC-8 monolayers were incubated for 48 h, then fixed with 8% formalin in PBS (pH 7.3) for 2 h at room temperature. After fixation, plates were blocked for 1 h with 1% bovine serum albumin (BSA) containing 0.002% Tween-20 in PBS, and they were then labelled for 30 min with rat polyclonal antibodies directed against C. parvum membrane proteins. A goat-anti-rat polyvalent antiserum conjugated with horseradish peroxidase was used in combination with a 3,3',5,5',-tetra-methyl-benzidine (TMB) substrate kit to check for colour development at abs 630 using a BioTek EL311s ELISA plate reader. Each plate contained four or more positive controls including paromomycin 200 µg/mL, which consistently inhibits parasite growth 6070% in this assay. Inconclusive data, i.e. those in which the optimal parasite densities deviated more than ±3 standard deviations from the mean, were excluded from the calculations. Cytotoxicity for host cells was evaluated by cell death and/or detachment from the tissue culture plastic, as well as by an MTT cytotoxicity assay as described previously.14
In vivo testing for anticryptosporidial activity
Experimental design
Mice deficient in T-cell receptor (TCR-
) are incapable of clearing C. parvum infections.18 Therefore, they are useful for screening lead compounds against cryptosporidiosis. Oocysts were isolated and purified from the faeces of calves experimentally inoculated with C. parvum as described previously.19 Neonatal TCR-
-deficient mice were infected by gavage with 1 x 103 C. parvum oocysts in 100 µL of 0.15 M PBS at 7 days of age. Mice were then treated either with PBS (controls) or test compounds beginning at 10 days of age. The drugs were administered orally by gavage twice daily for 6 or 7 days using a 24G animal feeding needle at a dose of 0.67 mg/kg body weight. Mice were euthanized at 21 days of age when intestinal biopsies were performed, and sections from them were examined both for the presence of C. parvum and for histopathological changes due to the infection.
Assessment of infection
Intestinal sections from the distal ileum and caecum were fixed in 10% formalin and embedded in paraffin. Histological sections (4 µm) were cut, stained with haematoxylineosin and examined microscopically both for intracellular stages of C. parvum and for pathological lesions due to the infection. Infectivity scores (IS) were determined as described previously.20,21 Briefly, a score of 0 means that no intracellular stages of C. parvum were detected; 1 means that a few enterocytes containing parasites were observed; and 2 means that many intestinal cells contained asexual, intracellular stages of C. parvum. Scores were determined by examining at least 100 fields in individual tissue sections. Means were calculated for each treatment group, and the data were expressed as group mean ± S.E.M.
Assay for cytopathic activity against host cells. Briefly, uninfected HCT-8 monolayers were incubated in several concentrations of experimental drugs for 48 h and were then exposed to 50 µL of medium containing 0.8 mg/mL sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis-(4-methoxy-6-nitro)benzene-sulphonic acid hydrate (XTT; Sigma) and 100 µM phenazine methosulphate (Sigma) for 1 h at 37°C, so that a change in colour could develop as described previously.21 The absorbance was read at 450 using a BioTek EL311s ELISA plate reader.
Statistical significance
Mean IS were analysed by one-way analysis of variance followed by TukeyKramer multiple comparison tests, or 2 x 2 contingency tables were formulated and the percentage infected analysed by Fishers Exact Test. Data were considered significant when P < 0.05.
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Results |
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In order to determine whether any of the active, non-toxic BIQs were also effective in vivo, one compound each with low, high and intermediate in vitro activity (12, 13 and 19, respectively) was administered orally to neonatal TCR--deficient mice that had been infected with C. parvum 3 days earlier. Anticryptosporidial activity for all three BIQs tested in vivo (Table 3) was comparable to that observed in vitro, i.e. BIQ 13 > 19 > 12. These BIQs significantly (P < 0.05) reduced the number of intracellular, asexual stages of C. parvum within epithelial cells of the caecum as measured by IS of 0.42, 0.20 and 0.63, respectively, whereas the IS of mock-treated mice was 1.44.
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Discussion |
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Depending upon the substitution pattern, non-alkylated compounds were either highly active (BIQ 2, 100%) or inactive (BIQ 1, <10%) at 25 µM. Interestingly, even minor modifications like an ethyl substitution at N1 (BIQs 1416) reduced the anticryptosporidial activity to <10%. Analysis of the BIQ rates of inhibition at 50 µM also showed that introducing an aromatic (BIQ 22) or cyclohexane (BIQ 23) ring on the parent structure drastically reduced activity to <10%. Taking a closer look at the substitution pattern on the aromatic ring revealed that hydroxy groups or halogenation at positions C-5 and C-8 seemed to contribute significantly to activity against C. parvum. Both peri-substitution on the aromatic ring and meta-substition of a methyl or hydroxy group lead to a marked loss in activity, e.g. at 50 µM the inhibition of growth by BIQs 7, 11 and 12 was <10%, 45% and 43%, respectively.
As mentioned previously, additional testing of selected BIQs in a TCR--deficient murine model of chronic cryptosporidiosis that mimics the disease in AIDS patients confirmed the efficacy observed in vitro. Remarkably, treatment with BIQ 13 resulted in a nearly complete cure of experimental mouse infections. There were no significant side effects at any dose tested, even including oral dosing at 169 µg/kg body weight.
As with simple naphthoquinones, the presence of redox groups may contribute to the considerable activity and low toxicity of BIQs against C. parvum and plant naphthoquinones against species of Leishmania and Plasmodium.12,22 By virtue of structural analogy to naphthoquinones and their mode of action, BIQs are postulated to inhibit parasite growth by causing disruption of mitochondrial electron transport,23 especially at sites III (bc1 complex) and IV (cytochrome c oxidase) where quinones are known to play a role.24,25 For instance, it is well documented that trypanosomatids can generate radicals from redox cycling of ortho-naphthoquinones26 and that naphthoquinones inhibit the consumption of oxygen by Leishmania species.27 Although the remarkable efficacy of BIQs for C. parvum both in vitro and in vivo was unknown, using the mitochondria-specific vital fluorescent dyes DIOC6, MitoTracker Green FM, Rhodamine B and Rhodamine 123 (Molecular Probes), we have recently shown that the relic mitochondrion of C. parvum28 possesses a membrane potential (mt) that can be disrupted by 1 nM KCN or 100 µM oligomycin (J. S. Keithly, unpublished results). Therefore, as in trypanosomatids and other apicomplexans,29,30 it is likely that some part of BIQ efficacy against C. parvum is related to mitochondrial function.
In contrast to the widely tested naphthoquinones, the introduction of an imidazole ring in BIQs seemed to be important for reducing toxicity. Therefore, in order to develop a safe and effective lead compound, the precise target and mechanism of action of this pharmacophore needs to be determined. We are currently testing lead BIQs for their ability to inhibit O2 uptake by sporozoites of C. parvum and to disrupt mt as measured by the dissipation of fluorescence of mitochondria-specific vital dyes. Similar methods have been used successfully both in vitro and in vivo to determine the mode of action of mitochondrial inhibitors against the apicomplexans Plasmodium yoelii and Toxoplasma gondii,29,30 as well as trypanosomatids.24 In the future, molecular modelling might also be useful for synthesizing more efficacious BIQs.
In conclusion, our study shows that BIQs exhibit interesting anticryptosporidial properties with low toxicity for mammalian host cells. These results may have implications for other intracellular apicomplexans like Plasmodium and Toxoplasma, as well as the kinetoplastids Leishmania and Trypanosoma. In vivo experiments in a murine model confirmed the in vitro results. Additional drug formulations must now be tested and experimental procedures varied to substantiate further and possibly improve the already appreciable antiparasitic activities of BIQs in vivo. The anticryptosporidial potential of the BIQs described here may well contribute to the search for new and selective therapies against cryptosporidiosis. This is especially important, because a safe and efficacious treatment for this AIDS-associated opportunistic disease is still not available.
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Footnotes |
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