Chemotherapeutic Effect of Arprinocid in Experimental Cryptosporidiosis

Abstract

More than 40 antimicrobial agents, including coccidiostats and other anti-protozoal compounds, broad-spectrum antibiotics, and anthelmintics, have been tested against Cryptosporidium in the course of treatment of infections in humans (Lasser et al., 1979, Human Pathology 10: 234-240; Weisburger et al., 1979, American Journal of Clinical Pathology 72: 473-478; Stemmermann et al., 1980, American Journal of Medicine 69: 637-642; Weinstein et al., 1981, Gastroenterology 81: 584-591; Sloper et al., 1982, Gut 23: 80-82; Moon et al., 1982, Veterinary Record 110: 181; Tzipori et al., 1982, Australian Journal of Experimental Biology and Medical Science 60: 187-190; Angus et al., 1984, Veterinary Record 114: 166-168). Most of these drugs were ineffective against Cryptosporidium. Of the drugs that have been prophylactically used in mice, arprinocid caused a marked reduction in oocyst excretion with no histological evidence of infection (Angus et al., 1984, loc. cit.), although it had been reported earlier to be ineffective in mice when given therapeutically after the inoculation of oocysts (Tzipori et al., 1982, loc. cit.). To date, arprinocid has not been used in humans. The inconclusive findings regarding drugs purported to be effective against cryptosporidiosis prompted the present investigation to test the efficacy of arprinocid, a drug with the greatest promise. Arprinocid, 6-amino-9-(2-chloro-6fluorobenzyl) purine, is an anticoccidial drug with activity against Eimeria (Miller et al., 1977, Poultry Science 56: 2039-2044; Wang et al., 1979, Biochemical Pharmacology 28: 2241-2248). It exerts its activity on the parasite by blocking hypoxanthine-guanine salvage. In addition, arprinocid also inhibits dihydrofolate reductase and glucose-6-phosphate dehydrogenase. Oocysts of Cryptosporidium were obtained from 1-day-old calves infected with 1 x 107 oocysts and feces collected from day 3 to day 7. Fecal samples were passed through a graded series of sieves and preserved in 2.5% K2Cr2O7. Oocysts were purified by passage through 2 aluminum sieves, ethyl acetate, 1% Clorox, and a discontinuous sucrose gradient. After washings in PBS, an aliquot of 0.02 ml of the sediment in 2.5 ml of PBS was used for smears, which were air dried, fixed (100% methanol), and stained with Ziehl Neelsen carbol fuchsin for 30 min