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FEMS Immunology and Medical Microbiology 26 (1999) 109^113 A comparison of the e¡ects of two dinitroanilines against Cryptosporidium parvum in vitro and in vivo in neonatal mice and rats A. Armson *, K. Sargent, L.M. MacDonald, M.P. Finn, R.C.A. Thompson, J.A. Reynoldson Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, W.A. 6150, Australia Received 5 July 1999 ; accepted 14 July 1999 Abstract The effects of two dinitroanilines, oryzalin and trifluralin, were compared against Cryptosporidium parvum, in vitro using HCT-8 cells and in vivo using neonatal Swiss ARC mice and Wistar neonatal rats. In vitro, oryzalin and trifluralin exhibited IC50 values (concentration necessary to cause a 50% inhibition) of 750 and 800 nM, respectively. A viability assay showed that neither compound produced a cytotoxic effect on the host cells at concentrations as high as 1 WM. The in vivo component of this study consisted of inoculation of neonatal mice and neonatal rats with 105 viable oocysts of C. parvum per animal and the subsequent treatment of this infection with trifluralin and oryzalin administered via gastric intubation. At doses of 100 mg kg31 body weight administered twice daily for 3 consecutive days, trifluralin had no statistically significant effect on the number of oocysts recovered from the gut of either rats or mice compared with controls, whereas at the same concentration, oryzalin caused 90 and 79% inhibition of oocysts recovered from mice and rats, respectively. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. 1. Introduction Cryptosporidium parvum is a widespread pathogen infecting domestic and wild animals as well as humans [1]. The infection causes diarrhoeal disease which is self limiting in immunocompetent hosts but can be life threatening in immunocompromised individuals such as those infected with human immunode¢ciency virus [2]. Cryptosporidiosis has been recognised as a signi¢cant public health threat with * Corresponding author. Fax: +61 (08) 9310 4144; E-mail: [email protected] the potential for waterborne outbreaks involving large numbers of people [3] and is also of considerable signi¢cance to the cattle industry, where it is the most common cause of neonatal calf diarrhoea and associated calf morbidity [1,4]. At present, there is no e¡ective treatment for cryptosporidiosis despite the fact that many compounds have been tested for e¤cacy against this parasite either prophylactically or as a means of therapy [5,6]. The dinitroanilines are tubulin-binding agents that were originally recognised for their herbicidal properties. More recently, a number of in vitro studies have compared the anti-cryptosporidial e¡ects of several dinitroanilines as well as dinitroaniline ana- 0928-8244 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 8 2 4 4 ( 9 9 ) 0 0 1 2 7 - 3 FEMSIM 1112 8-10-99 110 A. Armson et al. / FEMS Immunology and Medical Microbiology 26 (1999) 109^113 logues [7^9]. However, as far as we are aware, no previous studies have examined the in vivo e¤cacy of these compounds against C. parvum. In this study, we compared the in vitro and in vivo anti-cryptosporidial e¡ects of two dinitroanilines, tri£uralin and oryzalin, using neonatal mice and rats. Brie£y, this method involves counting 30 ¢elds of view which are randomly determined. The parasites counted in the 30 ¢elds of view were summed and the mean of duplicates was established. The percentage inhibition (Inh %) was determined by Inh % total parasites in control3total parasites in drug-treated well total parasites in control 2. Materials and methods 2.1. Puri¢cation, in vivo ampli¢cation and isolation of infective Cryptosporidium oocysts A bovine Cryptosporidium isolate was collected from calf faecal samples using phosphate-bu¡ered saline (PBS)-ether sedimentation and was washed as described by Morgan et al. [10]. In order to induce infectivity of the oocysts to cell culture it is necessary to passage them in mice. Therefore, the resuspended oocysts were inoculated into 7^8-day old Swiss ARC mice via gastric intubation of 10^12U104 oocysts per mouse using the method of Meloni and Thompson [11]. The mice were killed on day 7 post-infection and the oocysts processed as previously described [11]. Oocysts were further puri¢ed using a Ficoll gradient and subsequently counted on a haemocytometer. Oocysts were stored at 4³C in PBS containing ampicillin and streptomycin. 2.2. Infection of HCT-8 cells with infective oocysts and in vitro drug assay HCT-8 cells were grown to con£uence in 24-well plates in RPMI medium containing 10% foetal bovine serum. The cells were then rinsed in PBS, infected and then treated as described by Meloni and Thompson [11]. The appropriate drugs were then delivered in 5-Wl aliquots, in duplicate, dissolved in dimethyl sulfoxide (DMSO) to a ¢nal concentration of DMSO of no more than 0.5%. A range of drug concentrations was used in order to determine the concentration necessary to cause a 50% inhibition (IC50 ) of infecting oocysts. The cells were incubated for 72^120 h with constant exposure to the drugs or, in the case of the controls, vehicle alone. Cryptosporidium oocysts were then counted using a modi¢cation of the method of Meloni and Thompson [11]. U100 2.3. Measurement of cytotoxicity of drugs Cytotoxicity to host cells was determined using the CytoTox 960 kit (Promega) which measures lactate dehydrogenase as a guide to cell integrity. The assay was performed according to the manufacturer's instructions and both positive and negative controls were used. The assay was performed in quadruplicate. 2.4. In vivo analysis of drug activity against Cryptosporidium Stock solutions of drugs were made in 5-ml aliquots. Drugs were dissolved in 0.5 ml DMSO which was then added to 4.5 ml peanut oil using a modi¢cation of the method described by Fayer [12]. Neonatal mice and rats were infected with the oocysts in the manner described above. On day 4 post-infection, the animals were weighed and the mean weight for each litter was calculated. Using the mean weight, the dinitroaniline dose rate was determined such that each mouse or rat received 100 mg kg31 of drug in 100 Wl vehicle twice daily for 3 consecutive days. Controls received vehicle alone. Both treated and control mice and rats were killed on day 7 postinfection and processed in the manner described above [10]. For both drug treatments or control, three litters of either mice or rats were used and each litter was divided into three replicate groups giving a total of nine replicates. Mice replicate groups contained 2^3 animals whereas rat replicate groups contained two animals. Gut contents were pooled for each replicate group and the oocyst load was determined separately and average oocysts FEMSIM 1112 8-10-99 A. Armson et al. / FEMS Immunology and Medical Microbiology 26 (1999) 109^113 111 Table 1 E¡ect of dinitroanilines in vitro and in vivo on neonatal mice and rats infected with C. parvum compared with untreated controls Rat/mouse Rat Rat Mouse Mouse Drug Tri£uralin Oryzalin Tri£uralin Oryzalin Dose (mg kg31 ) 100 100 100 100 mg mg mg mg 31 kg kg31 kg31 kg31 % Inhibition IC50 value in vitro (nM) Nil 79 Nil 90 800 750 Neonatal mice and rats were treated 4 days post-infection with either vehicle, oryzalin or tri£uralin at 100 mg kg31 , administered at 12-h intervals (six doses). Oocysts recovered from treated and untreated animals were counted in order to determine the percentage inhibition. E¤cacies of the compounds were determined in vitro using HCT-8 cells infected with C. parvum and the oocyst yield was compared between treated and untreated controls. per mouse were calculated. The assignment of an animal to a replicate group within the treated litter was randomised using random numbers. Assays were performed three times for each drug and each concentration used. Statistical analysis was performed using a two-tailed paired t-test. 3. Results 3.1. In vitro studies The mean IC50 values for tri£uralin and oryzalin against Cryptosporidium in vitro were 800 and 750 nM, respectively (Table 1). At 800 nM, neither oryzalin nor tri£uralin caused a detectable increase in lactate dehydrogenase concentrations in the cell media using the CytoTox 960 kit. 3.2. In vivo studies Mean oocyst counts determined on treated and untreated animals demonstrated no signi¢cant e¡ect following tri£uralin treatment (six doses of 100 mg kg31 ) for either mice or rats (P v 0.05). In contrast, oryzalin caused 90% inhibition of oocyst numbers at the same dose rate for mice (P 9 0.001) and 79% inhibition for rats (Table 1) (P 9 0.005). 4. Discussion A safe and e¡ective anti-cryptosporidial agent has yet to be found. Previous studies have examined a number of compounds [6,8]. Yet, the most promising class of compounds remains the tubulin-binding di- nitroanilines. Like the benzimidazoles, the selective toxicity of the dinitroanilines is thought to be due to amino acid di¡erences in tubulin structure between the lower eukaryotes and mammals. These di¡erences are thought to account for the low mammalian toxicity (LD50 of 0.5 g kg31 for tri£uralin orally in 1^2-day old rats increasing to 36.5 g kg31 in 56-day old rats [13] and over 10 g kg31 orally for oryzalin in adult rats) [14], making this class of compounds a suitable starting point for the development of a treatment for cryptosporidiosis. The IC50 values of less than 1 WM achieved in the current in vitro study are similar to those previously reported [7] and represent achievable, albeit transient, concentrations within the gastro-intestinal tract. In fact, for an average neonatal mouse weighing 7 g, a prepared dose of 100 mg oryzalin or tri£uralin per kg body weight dissolved in 100 Wl solvent equals a concentration of approximately 50 WM. The concentration of dinitroaniline will naturally become more dilute in the gastro-intestinal tract. The comparable e¤cacies of tri£uralin and oryzalin in vitro are very di¡erent to their e¡ects in vivo in the strain of mice and rats used in this study. The total absence of any in vivo anti-cryptosporidial effect by tri£uralin at 100 mg kg31 body weight is disappointing and di¤cult to understand in view of the excellent e¤cacy achieved with an identical concentration of oryzalin. However, previous studies revealed that tri£uralin was rapidly cleared in rats by excretion in both faeces and urine [15]. Of the tri£uralin recovered from the faeces and urine in that study, only 10% comprised unchanged tri£uralin, suggesting that its metabolism may be the reason for the di¡erences in the in vivo e¡ects of the two drugs [15]. The rate of excretion was dose dependent FEMSIM 1112 8-10-99 112 A. Armson et al. / FEMS Immunology and Medical Microbiology 26 (1999) 109^113 with 80% clearance in 48 h following an oral dose of 1 mg kg31 , whereas the rate decreased to 60% clearance over the same interval at 10 mg kg31 . It could be reasonably postulated that the 100-mg kg31 dose used in the current study would slow the rate of clearance to an even greater extent. Oryzalin administered intraperitoneally to mice at 200 mg kg31 showed a peak plasma concentration of 25 Wg ml31 which decreased by half after 2.1 h and half again at 14.3 h. In contrast, when 300 mg kg31 was administered orally, the mean peak plasma level achieved was 4 Wg ml31 , suggesting poor absorption or ¢rst pass metabolism [14]. As with other gastro-intestinal parasites con¢ned to the mucosa, poor absorption may enhance rather than diminish e¤cacy. However, caution must be exercised when comparing the metabolism of di¡erent drugs in rats and mice where such di¡erences could be related more to animal than structural di¡erences of the compounds [16]. The partition coe¤cient (log P) and water solubility (ppm) values for oryzalin are 2.12 and 39 ppm, respectively, and for tri£uralin, they are 4.81 and 0.33 ppm, respectively. Taking into account the similarity of their structures coupled with the greater water solubility of oryzalin and its log P value of between 2^3, it is likely that oryzalin is more capable than tri£uralin of free movement through eukaryotic cells. Additionally, the milk diet of the neonatal mice and rats used in the current study and/or the peanut oil used as a vehicle for the administration of the compounds may act as a depot for tri£uralin, the less polar compound, thereby hindering its activity. The greater water solubility of oryzalin may prevent this scenario from occurring. Nonetheless, the unusual nature of Cryptosporidium in the host, being neither extracellular nor entirely intracellular, does add a further complication when predicting the e¡ects of solubility on the e¤cacy of a compound. Williams and Feil [17] examined the metabolism of tri£uralin in a rumen microbial culture and found that polar and non-polar metabolites were produced. Interestingly, only the polar metabolites were resistant to acid and base hydrolysis and acetylation, but were susceptible to reduction. Whether or not this resistance to hydrolysis and acetylation played a role in the sustained e¤cacy of oryzalin in the present study compared to tri£uralin is unknown and open to speculation. At present, we are using HPLC to ex- amine the metabolic changes that occur with these drugs in the murine model. The di¡erence in e¤cacy in vivo suggests that oryzalin rather than tri£uralin has the greater potential and would therefore be a better lead compound for the design of an e¡ective anti-cryptosporidial agent. Acknowledgements The authors would like to acknowledge the technical assistance and advice given by Dr. Wayne Best of the Chemistry Centre (WA). We would also like to acknowledge the continuing ¢nancial support we receive from SmithKline Beecham Pharmaceuticals and the Australian Research Council (ARC) in the form of a SPIRT research grant. References [1] Dubey, J.P., Speer, C.A. and Fayer, R. (1990) General biology of Cryptosporidium. In: Cryptosporidiosis of Man and Animals (Dubey, J.P., Speer, C.A. and Fayer, R., Eds.), pp. 1^29. CRC Press, Boston, MA. [2] Watzl, B., Huang, D.S., Alek, J., Darban, H., Jenkins, E.M. and Watson, R.R. (1993) Enhancement of resistance to C parvum by pooled bovine colostrum during murine retroviral infection. Am. J. Trop. Med. Hyg. 48, 519^523. [3] MacKenzie, W.R., Hoxie, N.J., Proctor, M.E., Gradus, S., Blair, K.A., Peterson, D.E., Kazmierczak, J.J., Addiss, D.J., Fox, K.R., Rose, J.P. and Davis, J.P. (1994) A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. New Engl. J. Med. 331, 161^ 167. [4] Xiao, L., Herd, R.P. and Rings, D.M. (1993) Concurrent infections of Giardia and Cryptosporidium in on two Ohio farms with calf diarrhoea. Vet. Parasitol. 51, 41^48. [5] Naciri, M., Mancassola, R., Rëpërant, J.M., Canivez, O., Quinque, B. and Yvorë, P. (1994) Treatment of experimental ovine cryptosporidiosis with ovine or bovine hyperimmune colostrum. Vet. Parasitol. 53, 173^190. [6] Woods, K.M., Nesterenko, M.V. and Upton, S.J. (1996) E¤cacy of 101 antimicrobials and other agents on the development of C. parvum in vitro. Ann. Trop. Med. Parasitol. 90, 603^615. [7] Arrowood, M.J., Mead, J.R., Xie, L.T. and You, X.D. (1996) In vitro anticryptosporidial activity of dinitroaniline herbicides. FEMS. Microbiol. Lett. 136, 245^249. [8] Armson, A., Meloni, B.P., Reynoldson, J.A. and Thompson, R.C.A. (1999) Assessment of drugs against Cryptosporidium parvum using a simple in vitro screening method. FEMS (in press). FEMSIM 1112 8-10-99 A. Armson et al. / FEMS Immunology and Medical Microbiology 26 (1999) 109^113 [9] Benbow, J.W., Bernberg, E.L., Korda, A. and Mead, J.R. (1998) Synthesis and evaluation of dinitroanilines for treatment of cryptosporidiosis. Antimicrob. Agents Chemother. 42, 339^343. [10] Morgan, U.M., Reynoldson, J.A. and Thompson, R.C.A. (1993) Activities of several benzimidazoles and tubulin inhibitors against Giardia spp. in vitro. Antimicrob. Agents Chemother. 37, 328^331. [11] Meloni, B.P. and Thompson, R.C.A. (1996) Simpli¢ed methods for obtaining puri¢ed oocysts from mice and for growing Cryptosporidium parvum in vitro. J. Parasitol. 82, 757^762. [12] Fayer, R. and Fetterer, R. (1995) Activity of benzimidazoles against cryptosporidiosis in neonatal BALB/c mice. J. Parasitol. 81, 794^795. [13] Budavari, S., O'Neil, M.J., Smith, A. and Heckelman, P.E. (1989) The Merck Index, 11th edn. Merck, Rathway, NJ. 113 [14] Dvorakova, K., Dorr, R.T., Gallegos, A., McClure, T. and Powis, G. (1997) Pharmacokinetic studies of the herbicide and anti-tumor compound oryzalin in mice. J. Chromatogr. B. 696, 275^281. [15] Erkog, F.U. and Menzer, R.E. (1985) Metabolism of tri£uralin in rats. J. Agric. Food Chem. 33, 1061^1070. [16] Reynoldson, J.A., Thompson, R.C.A. and Meloni, B.P. (1991) In vivo e¤cacy of albendazole against Giadia duodenalis in mice. Parasitol. Res. 77, 325^328. [17] Williams, P.P. and Feil, V.J. (1971) Identi¢cation of tri£uralin metabolites from rumen microbial cultures. E¡ect of tri£uralin on bacteria and protozoa. J. Agric. Food Chem. 19, 1198^ 1204. FEMSIM 1112 8-10-99