Metabolism of the schistosomicidal agent hycanthone by rats and rhesus monkeys*

Bull. Org. mond. Sante 1971, 45, 27-34Bull. Wid Hith Org. Metabolism of the Schistosomicidal Agent Hycanthone by Rats and Rhesus Monkeys* P. HERNANDEZ,1 E. W. DENNIS 2 & A. FARAH 3 The absorption, distribution, and excretion of hycanthone were studied with rats and rhesus monkeys using tritiated materials at the therapeutic dose recommended for man (3.0±0.5 mg/kg). Nine pairs of each species received single intramuscular doses of randomly tritiated (specific activity 193.7 mCi/mmol) hycanthone methanesulfonate and were then sacrificed at intervals between 15 min and 72 h after medication. Peak blood and tissue concentrations occurred 30-60 min after administration (plasma half-life-45 min). The highest concentrations were observed in the liver, spleen, kidneys and adrenals, but decreased rapidly (more than 80% of the dose was excreted in 48-72 hours). In monkeys a high concentration of the compound was found in the bile (hours 1-8), probably con- jugated to glucuronic acid. Radiochromatography showed only unchanged drug in the blood and tissues, except in the liver where rapid conversion occurred to sulfoxide in the rat and to the deethyl analogue in the monkey. The schistosomicidal activity of hycanthone in the mouse, the hamster, and the monkey has been des- cribed by Rosi et al. (1965), Berberian et al. (1967a), and Pellegrino et al. (1967). Berberian et al. (1967a) compared the oral and parenteral activity of hycanthone in experimental infections with Schistosoma mansoni and concluded that the schistosomicidal activity of a single intra- muscular administration was equivalent to that obtained by a 5-day regimen of oral medication. The investigations by Katz et al. (1968, 1969), Maritz (1968, 1970), and Clarke et al. (1969) demonstrated the effectiveness of hycanthone against Schistosoma haematobium and S. mansoni in man after daily oral doses (2.5±0.5 mg/kg) for 3-5 consecutive days or of a single intramuscular injection (3.0±0.5 mg/kg). The present investigation was performed in order to determine blood and tissue levels of tritiated hycanthone after a single intramuscular injection in rats and after a single intramuscular or oral dose in rhesus monkeys. The pattern of excretion and * From the Sterling-Winthrop Research Institute, Rensselaer, N.Y., USA. Research Pharmacologist. Director, Biology Division. ' Associate Director, Biology Division. species differences in the biological transformation of the compound are discussed. A report on a study of the uptake of tritiated hycanthone by male and female Schistosoma mansoni worms has already been published (Yarinsky, Hemandez & Dennis, 1970). MATERIALS AND METHODS Male Sprague-Dawley rats (Charles River) weigh- ing 250-300 g and male and female rhesus mon- keys weighing between 2 kg and 3 kg were used. Randomly tritiated hycanthone methanesulfonate, with a specific activity of 193.7 of mCi/mmol, was injected as a 10% solution into the semimembranous muscle in both species. For an additional study in monkeys the drug was administered by stomach tube followed by 2-3 ml of water. In all cases the dose was 3 mg base per kg of body weight. After administration of the drug, 9 pairs of rats were placed in metabolism cages. At time intervals of 1/2 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, 48 h, and 72 h, respectively, after medication, 2 animals were anaesthetized with ether, blood samples were drawn by cardiac puncture into heparinized syringes, and 2701 -27- P. HERNANDEZ, E. W. DENNIS & A. FARAH the tissues were removed and frozen immediately. Total urine and faeces were collected from the 24-, 48- and 72-hour groups. Monkeys were sacrificed by intravenous adminis- tration of pentobarbital after collection of blood in heparinized syringes. Those monkeys whose hycan- thone blood levels were followed for 72 h were maintained in restraining chairs and their urine and faeces were collected at 24-hour intervals. Food and water were given ad libitum. The tissues were weighed and homogenized in a Sorvall Omni-mixer with 9 volumes of 0.2 M sodium borate buffer, pH 9.5. An aliquot of the homogenate was extracted by shaking for 10 min with 5 volumes of dichloromethane. Blood and plasma were diluted with buffer and extracted as described for tissue homogenates. After centrifugation, an aliquot of the organic phase was evaporated to dryness under N2 in a counting vial and the residue was redissolved in 15 ml of a counting solution containing 7 g of PPO (2,5-diphenyloxazole), 0.3 g of POPOP (2,2'-p- phenylenebis(5-phenyloxazole, 100 g of naphthalene, 130 ml of bis(2 methoxyethyl ether, 33 ml of water and sufficient p-dioxane to make the volume up to 1 000 ml. Faeces required different processing because most of the radioactivity present was not extractable and the coloured extracts produced high quenching that resulted in low counting efficiency. Therefore, aliquots of faecal homogenates were burned in an oxygen atmosphere in a closed system, using a modification of the method of Kelly et al. (1961). After complete combustion the flasks were quantita- tively rinsed with 25 ml of a counting solution containing 6 g of PPO, 0.2 g of POPOP, 200 ml of methylcellosolve (2-methoxyethanol), and sufficient toluene to make the volume up to 1 000 ml. The combustion of some samples was carried out in a Packard (Model 300) Tri-Carb Sample Oxidizer according to the method of Kaartinen (1969). The composition of the counting solution used with the sample oxidizer was 3 g of PPO, 0.75 g of POPOP, 100 g of naphthalene, 150 ml of toluene, 40 ml of absolute ethanol, and sufficient dioxane to make the volume up to 1 000 ml. All samples were counted in a Packard Model 3003 Tri-Carb Scintillation Spectrometer. A total of 10 000 or more counts was obtained from each sample and the channel-ratios technique of Baillie (1960) was used for determination of efficiency. The recovery of added radioactive hycanthone from several tissues, blood, and plasma under the con- ditions described above varied between 85% and 103°0. The corresponding corrections were intro- duced in the calculations when applicable. In order to determine the nature of the radioactive compounds being detected in tissues, aliquots from the dichloromethane extracts were concentrated and spotted on 5 x 20 cm thin-layer chromatography plates. Bile was spotted directly and also after hydrolysis followed by extraction into dichloro- methane. For enzymatic hydrolysis, 100,ulitre of bile were diluted with 800 Mlitre of 0.1 N sodium acetate buffer pH 5.5 and hydrolysed overnight at 37°C with 10 000 units of ,B-glucuronidase and 5 000 units of sulfatase added as Glusulase (Endo). Sufficient non-radioactive hycanthone was placed on a side in all plates as a reference to visualize the location of radioactive hycanthone. The plates were developed in ether-methanol-triethylamine (80-10- 10) and scanned in a Packard Radiochromatogram Scanner Model 7201. RESULTS The distribution of the radioactivity as a function of time was measured in organs and tissues of medicated rats and monkeys and these data are presented in Tables 1 and 2 in terms of concentration of hycanthone base. For the purpose of comparison the absorption of hycanthone methanesulfonate after oral and intra- muscular administration of 3 mg of base per kg of body weight was studied in another group of rhesus monkeys. Two animals were used for each route of administration and 1 ml of blood was drawn at inter- vals. The levels of radioactivity found in blood ex- pressed in terms of hycanthone base per ml are shown in Fig. 1. The cumulative urinary and faecal excretion of the drug after intramuscular administration to rats and rhesus monkeys is shown in Table 3 and Fig. 2. The radioactivity excreted in monkey urine was further analysed to determine to what extent hycanthone and its metabolites are excreted as conjugates. To this end, an aliquot of the 0-24 hour urine was adjusted to pH 9.5 with 0.2 M borate buffer and shaken with dichloromethane, while another aliquot was first hydrolysed overnight with a mixture of fl-glucuronidase and sulfatase (Glusu- lase) and then extracted as described above. 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Absorption of 3Hhycanthnnthoe methnesulfondateciresu(X) monkeytotalexrtonwing the administatind ofaee 3c), of baeherumokgyofbodyoweingh eingler intramuscularlinetonororll mgprk fxoywiht)iue.i aete 0. i2 90 ,-1~ 100- x ui 70- ui 60- 0 ol 50 4 0 40 4 2 0 30Q4Lu 20- 0~~~~~~~~~~~~~0 HOURS POST-MEDICATION Fig. 2. Excretion of hycanthone in the urine (03) and faeces (X), and total excretion in the urine and faeces (OX, of rhesus monkeys following a single intramuscular injection of 3 mg per kg of body weight. Figures in parenthe- ses indicate the number of animals examined. 31 P. HERNANDEZ, E. W. DENNIS & A. FARAH Table 3 Cumulative urinary and faecal excretion (up to 72 hours after medication) of hycanthone and metabolites after a single intramuscular dose in rats and rhesus monkeys a % of dose % of dose Total % Animal excreted excreted of dose in urine in faeces excreted Rat 20.6 62.2 82.8 Rhesus monkey 46.7 36.0 82.7 a Dose = 3 mg base/kg. found that 35% of the radioactivity could be extracted before hydrolysis and that it corresponded mainly to free hycanthone. The remaining 65% could be extracted only after enzymatic hydrolysis and it was derived from conjugates of hycanthone, deethyl, hycanthone, and hycanthone sulfoxide. Fig. 3 shows the tracings obtained from the radio- chromatograms of the extractable radioactivity 0 z h101 A 0 I-. -i I- hi 0 J 30IW6 on1 U) ,4,"Itoi W0: 1 Fig. 3. Radiochromatographs obtained from extractable fraction of monkey urine before (A) and after (B) enzymatic hydrolysis. before hydrolysis (Fig. 3A) and after hydrolysis (Fig. 3B). It appears that in the 0-24 hour urine only hycanthone is present in the free form and that most of the radioactivity excreted can be accounted for as conjugates of the two main metabolites and traces of the deethyl sulfoxide. DISCUSSION The pattern of absorption and distribution of hycanthone in tissues and organs appears to be similar in both rats and monkeys (M. nziulatta). However, the levels of the compound found in blood and tissues are considerably higher in the imnonkey, particularly during the first 8 hours after medication. From the data presented in Tables 1 and 2 it can be concluded that in both species the peak levels are attained at about 30 min after the intramuscular administration of the compound, although con- siderable amounts are detected at 15 min. There is fast absorption during the first hour and almost RAT LIVER x 0 IL 0 W I 1\ 1 hi a 0 M IL Z 41 M U) hiZ MONKEY LIVER n~~~~~~~~. Z hi (- OX 0 co I 0 1h Fig. 4. Radiochromatographs from rat and monkey liver extracts obtained 30 min after intramuscular ad- ministration of 3 mg of tritiated hycanthone per kg. cz.-~Wi to 32 I METABOLISM OF HYCANTHONE BY RATS AND RHESUS MONKEYS complete absorption from the site of injection at the end of 72 hours. Thin-layer chromatography and scanning of the radioactive materials extracted showed that in both species radioactivity found in tissues and blood (Tables 1 and 2) corresponds to unchanged hycan- thone. However, liver extracts from rats and monkeys contained significant amounts of metabo- lites in addition to hycanthone (Fig. 4), whereas in monkey bile only conjugated hycanthone was detected (Fig. 5). The differences in the physiological disposition of the compound for the two species studied indicate that in the rat sulfoxidation is the major pathway and that in the monkey the main metabolite appears to be the deethyl derivative of hycanthone. These findings are in agreement with those of Rosi & Me- rola (unpublished data, Sterling-Winthrop Research Institute, 1964), cited by Berberian et al. (1967b), who also found the main metabolites formed in vitro to be the sulfoxide with rat liver preparations and deethyl hycanthone with monkey and human hepatic micro- somes. The evidence, therefore, suggests that the biological transformation of therapeutic doses of hycanthone in man would be similar to that observed in the monkey. Fig. 5. Radiochromatographs of extracts obtained be- fore (A) and after (B) hydrolysis of bile from monkeys that received 3 mg/kg of tritiated hycanthone. ACKNOWLEDGEMENTS The authors express their appreciation to Mr F. W. Gubitz for the preparation of the methanesulfonate salt of hycanthone and to Miss M. Palacios and Mr W. F. Banks for their excellent technical assistance. RESUME METABOLISME D'UN SCHISTOSOMICIDE, L'HYCANTHONE, CHEZ LE RAT ET LE SINGE RHtSUS On a etudie l'absorption, la repartition et l'excretion de l'hycanthone chez le rat et le singe rhesus apres admi- nistration du compose marqu6 au tritium a la dose thera- peutique recommandee chez l'homme (3,0±0,5 mg/kg). Neuf paires d'animaux de chaque espece ont requ une injection intramusculaire d'hycanthone marque, puis ont et6 sacrifi6es apres des intervalles de 15 minutes a 72 heures. Les concentrations maximales du produit dans le sang et les tissus ont ete enregistrees apres 30 a 60 minutes. Le foie, la rate, les reins et les glandes sur- renales presentaient les plus fortes teneurs, mais celles-ci se sont rapidement abaissees et plus de 80%o de la dose administr6e avait ete excr6t6e dans un d6lai de 48 a 72 heures. Chez les singes, l'hycanthone a ete d6cele a forte concentration dans la bile (apres 1 heure et 8 heures), probablement en conjugaison avec l'acide glucuronique. La radiochromatographie a montre que chez les deux especes l'hycanthone n'etait present dans le sang et les tissus que sous forme libre; cependant, le foie contenait une quantit6 notable d'hycanthone sous forme conjuguee. 33 34 P. HERNANDEZ, E. W. DENNIS & A. FARAH REFERENCES Baillie, L. A. (1960) Int. J. appl. Radiat., 8, 1 Berberian, D. A., Freele, J., Rosi, D. Dennis, E. W. & Archer, S. (1967a) Amer. J. trop. Med. Hyg., 16, 487-491 Berberian, D. A., Freele, H., Rosi, D., Dennis, E. W. & Archer, S. (1967b) J. Parasit., 53, 306-311 Clarke, V. de V., Blair, D. M. & Weber, M. C. (1969) Cent. Afr. J. Med., 15, 1-6 Kaartinen, N. (1969) Techn. Bull., Packard Instrument Company, No. 18, pp. 1-9 Katz, N., Pellegrino, J., Ferreira, M. T., Oliveira, C. A. & Dias, C. B. (1968) Amer. J. trop. Med. Hyg., 17, 743-746 Katz, N., Pellegrino, J. & Oliveira, C. A. (1969) Amer. J. trop. Med. Hyg., 18, 924-929 Kelly, R. G., Peets, E. A., Gordon, S. & Buyske, D. A. (1961) Analyt. Biochem., 2, 267-273 Maritz, J. C. (1968) in: Third South African Symposium on Infective Diseases, Department of Internal Medicine, University of Pretoria, 11 March 1968 (cited by: Schneider, J. (1969) Med. Proc., 14 June, p. 201). Maritz, J. C. (1970) S. Afr. med. J., 44, 126-128 Pellegrino, J., Katz, N. & Scherrer, J. F. (1967) J. Parasit., 53, 55-59 Rosi, D., Peruzzotti, G., Dennis, E. W., Berberian, D. A., Freele, H. & Archer, S. (1965) Nature (Lond.), 208, 1005-1006 Yarinsky, A., Hernandez, P. & Dennis, E. W. (1970) Bull. Wld Hlth Org., 42, 445-449