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Luna; («c.bfinfiiizfigsfif ...m Hm. . |vsz|l’.|. - \.. .41.“.— . b....\ F.” . 31...“! ...wl. ....f A ,Jll; CID,- ’vt' cynV Av it ‘ nvwwfiifidufllfi»!!! a 1’“), a!» u! zll . ...7¢.t|\ nl-il, ..ubvlurl ., n L . rot-l I}; I .!> _ 3.5.415}; TTTTTTTTTTT — S IIIIIIIIIIII ‘ 10 ummum:nmnmrummmnlnrrilmmlmum 19 ‘M H l 3 1293 00585 2003 F LIBRARY Michigan State L univCfliw . This is to certify that the dissertation entitled (ejacxmaz m 7%,; W W ”Z . bidt’cflom W51. ‘7 b” - I % cum¥w~fgej W 2 ‘ presente y Eur-yrs“? FQ/c/é—‘L 5M has been accepted towards fulfillment of the requirements for Pha‘ . degreein Phfirfi’lfiwloss’ Mid TOVM‘Co‘OSy 3M Majog'rofessor Date M r53 [(390 MS U is an Afliflnan've Action/Equal Opportunity Institution 0-12771 _,____ .___. ,. _ __ __ , ____J' PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before duo due. DATE DUE DATE DUE DATE DUE fl MSU Is An Affirmative ActiorVEqual Opportunity Institution REDUCTION IN THE RNA CONTENT OF SCHISTOSOMA MANSONI: A POTENTIAL MECHANISM FOR THE SCHISTOSOMICIDAL ACTION OF R0 15-5458 By Feleke Eshete A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1990 ti (0qu ABSTRACT Reduction in the RNA Content of Schistgsgma mansoni: A Potential Mechanism for the Schistosomicidal Action of Ro 15-5458 by Feleke Eshete Ro 15-5458 [lO-Z-(diethylamino)ethyl-9-acridanone(2-thiazolin-2- yl)hydrazone] has recently been shown by the Hoffmann-LaRoche & Co., Ltd. to be effective in rodents and baboons against the three principal species of schistosomes that cause the human disease, schistosomiasis. Apart from the chemotherapeutic evaluations of Ro 15-5458 against schistosome infections, no attempt has been made to pharmacologically characterize its actions and examine the effects of the compound on the parasites. The main objective of this thesis is to examine the effects of Ro 15-5458 on adult §ghistosoma mansoni after exposing parasites to the drug in vivo, with the intent of understanding the mechanism of its schistosomicidal action. In addition, the in vitro effects of the drug on the parasite, as well as its absorption kinetics, have been studied. in mm, no antischistosomal effect could be demonstrated when pairs of parasites isolated from mice were incubated for 72 h in a medium containing a 10 pH solution of the drug. However, the drug increased both the contractile activity and longitudinal tension of the parasite musculature. This effect of Ro 15-5458 was reversed by cholinomimetic Feleke Eshete agents and does not appear to be associated with the in yiyg schistosomicidal action of the drug. Administration of’a single dose (15 mg/kg) of Ro 15-5458 to infected mice resulted in the death of all parasites. The drug exhibited a slow onset of activity in yiyg; with the same dose, a significant dislodgment of worms from the mesenteric to hepatic portal veins (hepatic shift) was observed after a lapse of 4-5 days. Examination of parasites retrieved from mice dosed with Ro 15-5458 ‘(15 mg/kg) for drug-induced changes before the hepatic shift indicated no alterations in the surface membrane integrity, energy metabolism 'or erythrocyte consumption up to 3 days postdosing. However, this treatment resulted in a significantly reduced egg output, weight, protein content, and the incorporation of leucine into acid-insoluble fractions of parasites. These defects were preceded by a reduction in the total RNA content of the parasites. Quantitative hybridization of parasite RNA immobilized on nylon membranes with specific probes revealed that treatment reduced the amount of actin and superoxide dismutase mRNA and both the 18S and 285 rRNA content of the parasites. Administration of the same dose of Ro 15-5458 did not exert similar actions on treated but uninfected mouse liver RNA, suggesting selectivity in its effects. These results suggest that the schistosomicidal effect of Ro 15-5458 and/or its metabolic products is to inhibit the expression of parasite genes. It is postulated that the reduction in the RNA content of the parasite initiates a series of events which alters the host-parasite relationship and gradually results in parasite death. To my parents, without whose support, encouragement and sacrifice, this would not have been possible. ii ACKNOWLEDGEMENTS I gratefully acknowledge the generous support, patience and guidance of my advisor, Dr. James L. Bennett. The many opportunities I had in Dr. Bennett’s laboratory have contributed to my scientific development and future endeavors. I would also like to thank my committee members, Dr. Timothy G. Geary, Dr. Jeffrey F. Williams, Dr. Kenneth E. Moore and Dr. Theodore M. Brody for the gracious contributions of their time and knowledge in the completion of this work. Special thanks to Dr. Timothy G. Geary for his personal interest in my education and this project and Helen Cirrito for technical assistance. The financial support from the World Health Organization and the College of Osteopathic Medicine at MSU over the years was vital to the accomplishment of this thesis. TABLE OF CONTENTS Page LIST OF TABLES ............. - ................................... vii LIST OF FIGURES ............................................... ix CHAPTER I ..................................................... 1 General Introduction .................................... 1 A. Schistosoma mansoni ......................... 2 1. The parasite .......................... 2 2. Life cycle ............................ 5 3. The musculature ....................... 7 4. Intermediary metabolism ............... 9 a. Carbohydrate metabolism ......... 10 b. Lipid metabolism ................ 13 c. Amino acid and protein metabolism 13 d. Nucleotide metabolism and struc- ture of nucleic acids ........... 15 8. Pathology of Sghistgsgma mansoni infection .. 17 C. Control of Schistosomiasis .................. 18 D. Background and Objectives of Proposed Research .........., ......................... 20 Summary and Significance ................................ 22 CHAPTER II: The Absorption Kinetics of Ro 15-5458 ............ 24 Introduction ............................................ 24 Materials and Methods ................................... 24 A. Animal Studies .............................. 26 B. Determination of Ro 15-5458 in Plasma ....... 26 C. Mass Analysis ..... .......................... 27 D. Pharmacokinetic Analysis .................... 27 iv TABLE OF CONTENTS (continued) Results ................................................. A. Identification of Chromatographic Peaks ..... B. Elimination of Ro 15-5458 ................... C. Absorption of Ro 15-5458 .................... Discussion ... .......................................... Summary ................................................ CHAPTER III: The In Vitrg Effects of R0 15- 5458 on Adult Schistgsgma mansgn_ ............................ Introduction ........................................... Materials and Methods .................................. A. Infection and Parasite Recovery ............ B. In Vitrg Parasite Culture .................. C. Parasite Transfer .......................... D. Mechanical Activity Recordings ............. E. Egg Production Assay ....................... F. Uptake and Incorporation of Precursors ..... G. Incubation of Parasites in Drug-Activating System ..................................... 1. Source of liver ...................... 2. Preparation of liver homogenate frac- tion ................................. 3. Preparation of NADPH-Regenerating System ............................... 4. Incubation of Schistosomes with R0 15- 5458 ................................. H. Incubation of Parasites in Plasma Recovered. from Treated Rabbits ....................... Results ................................................ Discussion ............................................. Summary ................................................ CHAPTER IV: The Antischistosomal Effects of Ro 15-5458 Ad— ministration to the Mouse Host .................. Introduction ........................................... Materials and Methods .................................. A. Chemicals .................................. 28 28 35 35 41 51 52 72 74 74 .75 75 TABLE OF CONTENTS (Continued) Page 8. Drug Administration ........................ 76 C. Schistosomicidal Action of Ro 15-5458 ...... 76 D. In yitrg Culture and Parasite Transfer ..... 76 E. Measurement of Surface Membrane Changes and Muscular Activity .......................... 77 F. Measurement of Parasite Gut Pigment ........ 77 G. Determination of Parasite Height and Pro- tein Content ............................... 78 H. Determination of Glycogen, ATP, Lactate and Utilization of Glucose ..................... 78 I. Measurement of the Uptake of Metabolic Pre- cursors .................................... 79 J. Measurement of the Incorporation of Leucine into Parasite Protein ...................... 80 K. DNA and RNA Extraction ..................... 81 L. In Vitro Translatibn ....................... 82 M. Recombinant Plasmids and Probe Synthesis ... 82 N. Dot-Blot Analysis .......................... 83 0. Northern Blot Analysis ..................... 85 P. Southern Blot Analysis .................... 86 Results ................... . ............................. 86 Discussion ............................................. 122 Summary ................................................ 135 GENERAL DISCUSSION AND CONCLUSIONS ........................... 137 RECOMMENDED AREAS FOR FUTURE STUDIES ......................... 140 BIBLIOGRAPHY ................................................. 141 APPENDIX I ................................................... A1 APPENDIX II .................................................. A2 APPENDIX III ................................................. A3 APPENDIX IV ......................... ......................... A4 vi 10 LIST OF TABLES Antischistosomal activity against S. mansoni, S. nagm11991nn, 5. 11nnn1gnn in hamsters and mice ... Pharmacokinetic data from rabbits following a 50- mg/kg single oral dose of Ro 15-5458 ............. In 111:9 effect of R0 15- 5458 on lactic acid pro— duction by §. nnnsnn1 ........................... Effect of Ro 15-5458 on the contractile activity of adult paired S. nangnn1 and reversal by choli- nergic agonsits ................................. Effect of R0 15- 5458 on the longitudinal muscle tone of adult male S. man§0n1 and reversal by cholinergic agonists ............................ Effects of Ro 15-5458 and its analogs on the longitudinal muscle tone and contractility of adult S. mansoni ................................ The 1n vitrg effect of Ro 15-5458 on egg produc- tion by 5. nansonj .............................. Uptake and incorporation of radioactive precur- sors into adult 5. nnnsnni exposed to Ro 15-5458 1n 111:9 ........................................ Percent survival of parasites treated with 10 uM drug 1n 111:9 or after in yivo treatment with Ro 15-5458 .......................................... Effect of Ro 15-5458 on warm count in mice with hycanthone resistant strain of S. mansoni ....... vii 21 38 54 55 59 61 63 65 67 87 LIST OF TABLES (Continued) labia __q_Pa e 11 Survival of S. nnnsgnj 1n vigng after removal of parasites from infected mice treated with a 15- mg/kg single dose of Ro 15-5458 ................. 89 12 Effects of Ro 15-5458 on the muscular activity and surface membrane potential of adult male S. mgn§Qn1 3 days after dosing ..................... 90 13 Effects of Ro 15-5458 on gut pigment, glycogen and ATP content of S. mansoni ................... 95 14 Effect of Ro 15-5458 on carbohydrate utilization and lactic acid production of S. nansnn1 3 days after dosing .................................... 96 15 Effect of Ro 15-5458 on the protein content and weight of S. mnn§nn1 ............................ 98 16 Effect of Ro 15-5458 on the uptake of nucleic acid and protein precursors of S. mansoni re- covered 3 days after dosing ..................... 99 l7 7 In vjtrg translation of RNA isolated from S. nnnsnn1 ......................................... 105 18 The effect of a 15-mg/kg single dose of R0 15- 5458 on the total RNA content of S. mansoni ..... 110 viii Elam 10 ll 12 LIST OF FIGURES Drawing illustrating the external characteristics of male and female S. mansoni .................... Life cycle of S. nnnsnni ......................... Ro 15-5458 and Ro 15-9895 ........................ Mass spectrum of Ro 15-9895, Ro 15-5458 and its hydrolysis product .............................. Chromatograms of Ro 15-5458 and Ro 15-9895 ....... Average Ro 15-5458 concentration in plasma (log scale) following a SO-mg/kg oral dose ............ Plasma levels of Ro 15-5458 as a function of time following the oral administration of 50-mg/kg single dose to rabbits ........................... Average concentration of Ro 15-5458 in plasma of rabbits after a 50-mg/kg single oral dose ........ Schematic representation of the apparatus used to record the longitudinal muscle tension in adult male S. nnngnn1 ................................. Effect of Ro 15-5458 on contractile activity of paired S. mansgni 1n vigno ...................... Dose-response relationship between Ro 15-5458 and muscle tension of adult male S. mansoni ......... Effect of 20 uM Ro 15-5458, atropine and SHT on the longitudinal muscle tone of adult male S1 mansnn1 ......................................... ix 25 28 33 34 36 37 47 53 56 58 LIST OF FIGURE (Continued) Lianne 13 14 15 16 17 18 19 20 21 22 23 24 Effect of Ro 15-5458 on the response of schisto- some longitudinal muscle tension to carbachol ... Recombinant plamids .......... .................... Scanning electron micrographs of the dorsal surf- ace of male S. nnngnn1 after dosing the host ..... Effect of 1n ijg administered Ro 15-5458 on the in 111nn egg production by S. nansgnj ........... Effect of Ro 15-5458 on the incorporation of tritiated precursors into the total acid—insoluble fractions of S. mansgnj ......................... Effect of Ro 15-5458 on the incorporation [3H]- leucine into the 100K pellet proteins of S. mansoni ......................................... Time course of the incorporation of [”S]methio- nine into proteins in translation system after the addition of schistosome or mouse liver RNA ...... Fluorogram from the 1n v11ng translation of poly(A’) RNA .................................... Fluorogram showing the polypeptide pattern of the 1n vignn translation products of schistosome and liver RNA before and after treatment with Ro 15-5458 ................... . ...................... Densitometric scans from fluorograms of transla- tion products ................................... Effect of Ro 15-5458 treatment on the actin mRNA content of S. mansoni ........................... Effect of Ro 15-5458 treatment on the 18S and 28S rRNA levels of S. nansonj ....................... 60 84 92 94 100 103 104 107 108 109 112 113 LIST OF FIGURES (Continued) figure 25 26 27 28 29 30 31 Ethidium bromide-stained RNA gel ................ Effect of a IS-mg/kg dose of Ro 15-5458 and Ro 21-6787 on the actin mRNA content of S. mansonj . Effect of Ro 15-5458 administration on the two sizes of S. nangnni actin mRNA .................. Effect of Ro 15-5458 administration on the SOD mRNA content of S. mansgnj ...................... Effect of Ro 15-5458 administration on the 185 rRNA level of S. mansoni' ........................ EcoRI restriction analysis pattern of DNA isolated from R0 15- 5458- treated S,. nnngnn1 .............. Hind III restriction pattern of DNA isolated from R0 15 5458- treated S. n_n§nn1 ................... xi 115 117 119 120 121 123 125 CHAPTER I GENERAL INTRODUCTION It is estimated that more than 200 million people suffer from schistosomiasis (bilharziasis) and about three times this number are threatened with the infection (Iarotski & Davis, 1981; WHO, 1985). The disease is endemic in about 76 countries. It is most widespread in tropical and subtropical areas of Africa, South America, the Caribbean Islands, the Eastern Mediterranean and the Arabian Peninsula and South- East Asia. It is essentially an infection of rural and agricultural areas, where there exists poverty, ignorance, poor housing and substandard hygienic practices. Schistosomiasis ranks second only to malaria in terms of socioeconomic and public health importance. Schistosomiasis is caused by digenetic trematodes of the genus Sgn1§1n§nna, among which S1,nan§nn1, S. haemaLQbium, and S. japonicnm are the principal causative agents of human disease. Humans are exposed to the infective form of the parasite in water during occupational or recreational activities; agricultural work in irrigation schemes constitutes one of the schistosomiasis risks (Hunter, 1982; Kloos, I985; HHO ,1985). In most endemic areas, children 6-15 years of age have been identified as the most highly exposed and heavily infected age group and also contribute most to water contamination (Dalton & Pole, 1978; Kloos e1 31., 1983). Once the invasive form of the parasite finds its way into the ultimate host, the resulting larvae migrate until the parasites grow 2 and sexually mature. The male and female worms copulate and, depending on the species, inhabit the mesenteric and intra-hepatic veins or the vesical plexus, where they live for many years producing numerous eggs. Different species of schistosomes differ in geographical distribution, snail-host infectivity, response to drugs, pathogenicity and immunogeni- city. The pathology of schistosome infection involves many organs and is mainly due to a vigorous host response against the parasite eggs. A. Sgnjsgnsnna mansonj l. ' 1.119.933.3111 The blood fluke S. mangnni is a sexually dimorphic plathy— helminth. The adult worms (male 10-15 mm long and female 15-20 mm long) often live in copula, the female residing within the gynecophoral canal of the male (Figure 1). Parasites attach to the inner wall of the veins of the host through the ventral sucker, a muscular organ capable of sustained contraction. The digestive system of the parasite is well developed. The oral sucker of both male and female S. ngnsnn1 parasites leads into the cecum, through a non-muscular esophagus. The cecal wall consists of a syncytial lining epithelium (gastrodermis). Morphological evidence suggests that the gastrodermis is both secretory and absorptive in function. The gastrodermis cells secrete digestive enzymes, such as hemoglobinase, which break down hemoglobin from the host erythrocytes into a pigment which imparts a brown appearance to the parasites. Figure 1. Drawing illustrating the external characteristics of male and femaie S. 11131152111. OS, oral sucker; VS, ventral sucker; GC, gynecophoral cana . 4 The external surface (tegument) of adult schistosomes is a syncytial interface between the parasite and the host environment. It is an unusual double bilayer structure (McLaren & Hockley, 1977), consisting of an inner bilayer or plasma membrane overlaid by a secreted outer bilayer of lipid rich material (Hilson & Barnes, 1974). The tegument develops within 3 h of host penetration by the cercaria and is thought to be an adaptation to the host environment, enabling the parasite to evade the immune response of the host (Hockley a McLaren, 1973; McLaren, 1984; McLaren 11 11., 1975). The surface membrane of the parasite is in a state of rapid turnover with a continuous synthesis and degradation of membrane proteins (Kusel & Mackenzie, 1975; Tavares 11 11., 1980; Dean and Podesta, 1984) and rapid turnover of membrane lipids (Meyer 11 11., 1970, Vial 11 11., I985). The rate at which the membrane turns over (t",) has been reported to be in the range of 5 to 45 h by different investigators (Wilson 81 Barnes, 1977; Ruppel & McLaren, 1986; Saunders 11 11., 1987). Surface topography of the tegument reveals features consistent with an absorptive function (Hockley, 1973). During short-term 1n vitro incubations, the tegument appears to be the primary site involved in absorption of low molecular weight solutes such as glucose (Rogers & Bueding, 1975; Uglem & Read, 1975; Mercer & Chappell, 1986a), amino acids (Chappell, 1974; Asch & Read, 1975a,b; Isserof 11 11., 1976), pyrimidine and purine bases (Levy & Read, 1975a,b; Mercer & Chappell, I986b) and cholesterol (Haseeb 11 11., 1985). Hork by Fetterer, Pax & Bennett (I980a) has demonstrated that a well-defined tegumental potential of about -60 mV exists at the dorsal 5 surface of the male parasite that can be altered by changing physical or chemical qualities of the parasite environment in 11111. These investi- gators also predicted that a NafldckATPase pump may be operating and that it maintains an ion gradient across the parasite tegument (Fetterer 11 11., I980b). The worms feed on host erythrocytes, utilizing at least part of the hemoglobin molecule (Zussman 11 11., 1970); the female parasites ingest about 13 times more erythrocytes at a rate 9 times faster than males (Lawrence, 1973). This large requirement for blood cells by the female parasites is necessary to supply precursors for proteins and perhaps nucleic acids in egg production, which is estimated to be as high as 1100 eggs/female/day (Damian & Chapman, 1983). Although the specific roles of the gut and the tegument in the nutrition of these parasites is not understood due to technical limitations, parasites may also absorb solutes from the host body through the oral route and/or the tegument. 2. We The life cycle of the parasite involves three biochemically and.morphologically distinct forms of the parasite, snail and a vertebrate host (Figure 2). Some of the eggs deposited by the females into the bloodstream of the vertebrate host pass through the venule walls, cross the intestinal mucosa, reach the lumen, and are evacuated with the fecal material. Hhen these eggs come in contact with fresh water, a freely swimming form (miracidium) is released. This form penetrates an appro- priate snail host, asexually multiplies in this host and is transformed into a new infectious form, the cercariae. The male and female cercariae shed by the snail penetrate the skin of man or another vertebrate host. In the skin they change into schistosomula and travel in the lymphatic MAMMAL $112511 WATER egg (in feces) / \ ‘ miracidium ( inc-living) paired adults 1 ( mesenteries T sporocysts @1111 snails) 1 long stage Z£'\¢hisiosoinulum cercaria 6% 511m. (free-11111119 1 skin siege on schistosomuium Figure 2. Life cycle of S. m. 7 system to the right heart and lungs, from*which they migrate to thé portal circulation. They grow and sexually mature in the liver and descend to the mesenteric veins where they live for as long as 30 years actively producing eggs (Faust 11 11, 1934). .7 3. 1h1 ng§cnlatnr1 The musculature of S1 n1n11n1 is located immediately beneath the inner membrane of the tegument and consists primarily of an outer circular and inner longitudinal muscle layer. In the male schistosome, in which both sets of muscles are well developed compared to the female, the longitudinal muscle is important in locomotor activity, while the circular muscle is essential for the formation of the gynecophoral canal and thus maintenance of male-female pairing. The contractile elements of the myofibrils consist of thick myofilaments (18-40 nm diameter), each surrounded by an irregular array of 8-14 thin filaments (5 nm diameter) (Silk & Spence, I969). The ratio of thin to thick filaments and absence of transverse tubules and microtubules makes the muscles of the worm resemble vertebrate smooth muscles. The sarcoplasmic reticulum is poorly developed or absent, but rough elements can be found scattered; mitochon- dria appear in sac-like distentions of the sarcoplasm along myofibril bundles. The nuclei are located deeper than the muscle fiber bundles and are connected to them through cytoplasmic processes. Lipid globules as well as a- and fi-glycogen particles are distributed throughout the peripheral cytoplasm of muscle cells (Silk & Spence, 1969). The spontaneous motor activity of the parasite can directly be recorded using suction pipets in circuit with a force transducer (Fetterer 11 11., 1977). The technique has also been employed in the 8 measurement of the effects of various pharmacological agents and inorganic ions on the neuromuscular system of the parasites. High K’ (60 mM) induces contractions of the muscle, by depolarization of the muscle membrane thereby allowing the entry of Ca”; osmolarity greater than 300 mOSm, pH less than 6.8, and the concentration of inorganic ions below or above that found in Hanks’ balanced salt solution reduced the contraction rate of the male schistosome muscle (Fetterer 11 11., 1978; l980b). The motor activity of these parasites appears to be modulated and coordinated by the nervous system through the action of neurotransmit- ters. There is evidence suggesting that 5-hydroxytryptamine (5-HT) functions as an excitatory neurotransmitter, causing an increase in motor activity (Barker 11 11., 1966; Tomosky 11 11., 1974; Fetterer 11 11., 1977; Hillcockson & Hillman, 1984). Acetylcholine is believed to function as an inhibitory neurotransmitter, causing flaccid paralysis (Hillman & Senft, 1973; Barker 11 11.,1966; Fetterer 11 11., 1977). Catecholamines have also been implicated as putative neurotransmitters (Tomosky, Bennett & Bueding, 1974). A report by Pax 11 11. (1984) suggests the possibility that the neurotransmitters described above affect the longitudinal and circular'muscles of the parasite differentially; Only longitudinal muscle appears to possess cholinergic receptors, while only circular muscle appears to possess dopaminergic receptors. 5-HT receptors appear to be associated with both longitudinal and.circular'muscle function. Carbachol and the cholinesterase inhibitors physostigmine and metrifonate block electrically-induced contractions of the longitudinal muscle (Pax 11 11., I981). The muscarinic receptor antagonist atropine and the antischisto- somal agents hycanthone and oxamniquine induce a marked increase in 9 contractile activity and were observed to reverse the paralytic effect of carbachol on parasite musculature (Hillman & Senft, 1975; Pica-Mattoccia & Cioli, 1986). 4- 1mm!!! More important data concerning intermediary metabolism has been collected for S. n1n11n1 than any other schistosome species because of its widespread geographical distribution and the simplicity of propagating the parasite in the laboratory. Nevertheless, knowledge on the metabolism of the parasite is far from complete; more research is needed, especially on the anabolic pathways of the parasite. In addition, most of the information on the metabolism of these parasites is derived from experiments carried out i_n 111:1. Hhether this information relates to the situation 1n 11111 has remained controversial, because several reports (Floyd & Nollen, 1977; Shaw & Erasmus, 1977; Fried, 1978; Basch & Humbert, 1981) indicate that systems employed for the maintenance of adult schistosomes 1n 11111 are inadequate. Hhile lengthy survival periods 1n vitro have been reported for adult.S, m1n11n1 (Robinson, 1956; Lancastre &.Golvan, 1973), deterioration of'worms during culture has been observed. Ultrastructural studies on the reproductive system reveal that the testes (Floyd & Nollen, 1977), and the vitelline gland and ovary (Floyd & Nollen, 1977; Shaw & Erasmus, 1977) of S_. 1111115911 degenerate comencing S to 6 days after isolation from the host. Egg production invariably ceases within 2-3 weeks in culture (Michaels & Prata, 1968; Newport &.Heller, 1982) and reduction in parasite size, glycogen and protein content has also been observed (Zussman 11 11., 1970; Basch & Humbert, 1981; Mercer & Chappell, 1985a). 10 a; gannnnydr1te n1tannli§n Adult S1 n1n§nn1 obtain the major portion of their energy from glucose. Glucose is taken up primarily through the tegument by mediated transport and simple diffusion (Rogers & Bueding, 1975; Uglem & Read, 1975; Mercer & Chappell, I986a). The transfer of glucose from male to female parasites has also been observed (Cornford & Huot, 1981). Other hexoses such as galactose (Isseroff, Bonta & Levy, 1972), fructose and mannose (Bruce 11 11., 1974) are also taken up through the tegument. The pioneering work of Bueding (1950) suggested that 1n 11111, the adult parasites consume an amount of glucose equivalent to 20- 30% of their dry weight in an hour, converting 80% of the glucose to lactic acid. Glycolysis was then identified to be the main pathway for energy extraction in these parasites. In a more recent study, the same group concluded that glucose utilization, lactic acid production and ATP levels were the same under aerobic and anaerobic conditions and all the ingested glucose and endogenous glycogen are quantitatively metabolized to lactic acid (Bueding & Fisher, 1982). The original work by Bueding (1950), on the other hand, demonstrated the consumption of oxygen by schistosomes. The 0, uptake was increased by inclusion of glucose in the medium and a difference in the respiratory quotients was observed in the presence and absence of glucose in the medium, suggesting the ability of these parasites to oxidize glucose to water and C0,. However, in a complete state of inhibition of parasite respiration with cyanine dyes, the rate of glycolysis was not affected and no adverse effects on the parasites were observed. Thus, it was concluded that S. nansnnj, are homolactic fermenters and derive their ATP solely through this pathway. 11 The role of 02 in these parasites is not well understood yet, although the parasites live in an aerobic environment. Recent studies suggest that schistosomes, although relaying heavily on glycolysis for'ATP synthesis, generally have wider metabolic capabilities with regard to oxidative pathways. Although glycolysis appears to be the major pathway of glucose metabolism, recent studies indicate that no stochiometric relationship exists between the amount of glucose utilized and lactic acid excreted (Rahman, Mettrick & Podesta, 1985a; McManus, 1986). For instance during 6 h aerobic incubations (McManus, 1986) paired and female S1 m1n§nn1 produced glucosezlactate ratios of approximately 1:1, while for male parasites the ratio was 1:2, suggesting that the females were channelling a substantial amount of' glucose into the synthesis of mucopolysaccharides, glycoproteins and glycolipids (McManus, 1986; Rahman, Mettrick & Podesta, 1985b). In addition, Van Oordt 11 11. (1985) incubated parasites in simple salt solution with [6-“C]-glucose and determined C02 produced. Subsequent calculations indicated that at least one third of the energy production of adult schistosomes occurs by aerobic processes. However, in addition to using a physiologically stressful medium, they assumed in their calculations that the tricarboxylic acid (TCA) cycle and coupled electron transport chain operate in schistosomes as efficiently as in mammalian tissues, which may not be the case. Ambiguities prevail in the literature about the existence of a functional TCA cycle, since enzymes and intermediates have not been completely demonstrated (Coles, 1972; Smith 81 Brown, 1977; McManus, 1986). S. mngnn1 produce CO2 from glucose, fructose and mannose (Bruce 11 11., 1974; Van Oordt 11 11 ., 1985). 12 However, the pathway that produces the C02 has been assumed to be the TCA cycle but has not been identified. The existence of an electron transport system and individual cytochromes (b, c,, c, a/a,) have been reported (McManus, 1986), which indicates the potential to carry out oxidative phosphorylation. However, there is no study that explains how glycolysis and the electron transport system are coupled in schistosomes. Another possible source of energy for S.na_r_1_s_q_n_1 is endogenous glycogen. Glycogen constitutes about 15% of the dry weight of adult male schistosomes, while the equivalent figure is about 5% for the females (Bueding & Koletsky, I950; Lennox & Schiller, 1972). The function of the glycogen reserve in these parasites is not well understood. Nonetheless, it may serve as a transient source of energy (Mercer 81 Chappell, 1985a; Tielens & Van den Bergh, 1987). In addition, schisto- somes convert glycogen into lactic acid when parasites are incubated in glucose free medium (Bueding, 1950; Bueding 81 Fisher, 1982). Rapid synthesis and degradation of glycogen has also been shown 1n 1111 after administration of radiolabeled glucose to infected hamsters (Tielens 11 11., 1989a). Parasites, maintained 1n 111:1, can synthesize glycogen from other precursors, such as fructose and mannose (Tielens 11 11., 1989b). The relevance of these studies to glycogen metabolism 1n 1111 is not clear since parasites were incubated in sugar concentrations higher than they would encounter in the host. _I_n v_j_t_r_q studies on the synthesis and degradation of glycogen suggest that, at glucose concentrations near the physiological levels of the human host, parasites rapidly deplete their 13 glycogen reserve despite increased uptake and incorporation of glucose into glycogen (Bueding, 1950; Mercer & Chappell, 1985a, 1986a). b. ' id l s Studies on the lipid composition of adult S. n1n11n1 reveal that schistosomes contain significant amounts of phospholipids, triglycerides, and cholesterol as free sterol, as well as small amounts of cholesterol esters and free fatty acids (Smith & Brooks, 1969; Meyer 11 11., 1970). Essentially all the phospholipids in these parasites are in the form of glycerophospholipids. Phosphatidyl choline (39%) is the major phospholipid, along with smaller amounts of phosphatidyl ethanol- amine (3%). The synthesis of both phospholipids is thought to start with choline (Young 81 Podesta, 1985). In addition traces of phosphatidyl serine, phosphatidyl inositol and cardiolipin have been detected in these parasites (Meyer 11 11., 1970). Schistosomes are host-dependent for sterols and long chain fatty acids, although possessing the capacity to elongate exogenous- ly supplied fatty acids (Smith 11 11., 1970; Meyer 11 _1., 1970). They incorporate [l-“Clacetate, uniformly labeled [“C]glucose and [1-“C]oleate into their triglycerides and phospholipids. Acetate is incorporated into preformed fatty acids while most of the glucose ends up in the composition of the glycerol backbone of triglycerides and phospholipids. c. Aninn 1111 1ng nnn1ein m1taboli§n Little is known about the amino acid and protein metabolism of schistosomes. The small amount of data accumulated in the literature is restricted mainly to uptake and/or incorporation studies (Chappell, 1974; Asch & Read, l975a,b; Isserof 11 11., 1976; Chappell & 14 Walker, 1982; Mercer & Chappell, 1985b) and the amino acid composition of total proteins (Robinson, 1961; Senft 11_11., 1972; Chappell & Walker, 1982). These studies indicate that, 1n 111:1, amino acids enter through the tegument by diffusion and mediated transport. The uptake of amino acids varied depending on the sex and pairing state of the parasites. Tyrosine is selectively taken up by the female vitelline cells (Erasmus, 1975). Leucine uptake rates were higher for separated parasites compared to pairs (Mercer & Chappell, 1985b). Limited work has been reported regarding the intercon- version and the synthesis of schistosome amino acids using carbon skeletons derived from glucose. A small amount of glucose is converted to amino acids (ala,asp, glu), and only 5 amino acids (ala, arg, asp, gly, ser) were converted to other amino acids. The interconversion is thought to have little significance to schistosome protein synthesis, except for glutamine (from alanine) and proline (from arginine), which were incor- porated into proteins (Chappell & walker, 1982). In addition, histidine has been shown to be metabolized via decarboxylation, deamination and transamination reactions to products including histamine and glutamate (Saber & Wu, 1985). Transamination (Garson & Williams, 1957; Chappell & Walker, 1982) and decarboxylation (Bruce 11 11., 1972; Foster 11 11., 1989) reactions have also been examined, but only limited attempts to elucidate pathways of amino acid catabolism and understanding the fate of the products have been made. Biosynthetic processes in the adult parasite must proceed at a high rate, since schistosomes produce enormous amounts of eggs and are known to rapidly renew their surface membrane. Thus, an 15 active protein synthetic mechanism must operate in these parasites. However, the mechanism of protein synthesis and the characteristics of enzymes involved in the process remain completely unstudied in schisto- somes. Messenger RNA has been isolated and translated 1n 111ng_in a rabbit reticulocyte system to products that react with the host imune sera (Tenniswood & Simpson, 1982; Taylor 11 11., 1983; Knight 11 11., 1984). A parasite>derived, cell-free protein synthesizing system has also been reported (Lukacs 11 11., 1980). d. ' ° r n ic a ’ Nucleic acids are essential components of all living organisms. The building blocks for these macromolecules are both purine and pyrimidine nucleotides. Most mammalian cells have the ability to synthesize purine and pyrimidine bases 11 nnxn. Preformed bases as well as nucleosides can also be converted to nucleotides by salvage routes. Pathways for salvage and the 11 mg synthesis of pyrimidines have been shown for S, n1n1nn1 (Hill 11_11., 1981; Iltzsch 11 11., 1984). Activities of all of the enzymes involved in the 11 nnxn synthesis of uridylic acid (UMP) have been identified in extracts of the parasites (Aoki & Oya, 1979; Hill 11 11., 1981; el Kouni 11 11., 1983). Enzymes of pyrimidine salvage pathways have also been identified (Senft 11 11., 1973; el Kouni 11 11. 1983). Activities of both thymidine and deoxycytidine kinases were present. However, parasite extracts have no uridine kinase or thymidine phosphorylase but contain a uridine phosphory- lase. The preferred substrate for uridine phosphorylase is uridine; the enzyme also catalyzes the reversible phosphorolysis of deoxyuridine, and 16 deoxythymidine, but not cytidine, deoxycytidine, or orotidine (el Kouni 11 11., 1988). S. nnngnni, on the other hand, lacks 11 nnxn purine biosynthesis mechanisms (Senft & Crabtree, 1983) and so rely on the host for the supply of preformed purines for nucleotide synthesis in the salvage pathways. Studies using intact S, n1n§nn1 revealed that adenosine was primarily deaminated by adenosine deaminase and that the inosine so formed could be salvaged to adenine nucleotides by sequential actions of purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase, adenylosuccinate synthetase and adenylosuccinate lyase (Senft, Senft & Meich, 1973; Senft 11 11., 1973). mm uptake studies (Levy & Read, 1975a,b) indicated that both purine and pyrimidine bases enter the parasite through the tegument. They postulated that adenine, guanine, hypoxanthine, adenosine and uridine entered by diffusion and mediated transport through distinct sites. In contrast, the uptake of cytosine, thymine and uracil appeared to be by diffusion. Since these parasites consume large numbers of host erythrocytes, substrates for salvage mechanisms may also be contributed by these cells. Studies by Simpson, Sher 81 McCutchan (1982) demonstrated that the haploid genome size of S. n1n11n1 is about 2.7x10° base pairs, which is about a tenth of the size of mammalian species. Base composition studies of the parasite genome from buoyant density'measurements (Hillyer, 1974) and a codon frequency table generated from published data (Meadows & Simpson, 1989), indicate about 34.3% G-C and a high A-T content (66%). The genome contains no detectable amounts of modified base pairs and has 17 both repetitive and unique sequences of DNA as in other eukaryotic organisms (Simpson, Sher & McCutchan, 1982). The highly repeated sequences were constituted mostly by the genes coding the two large ribosomal RNA (rRNA) molecules. The structural organization of the rRNA genes is similar to other eukaryotic organisms and they are encoded within 10 kb tandem repeats (Simpson 11 11,, 1984; Van Keulen 11 11., 1985). Both class sizes of rRNA molecules have a sedimentation coefficient similar to other eukaryotic species. However, under denaturing conditions the 28S rRNA splits into two equal sized molecules about the size of the 185 rRNA suggesting, the existence of a nick in the large rRNA (Tenniswood & Simpson, 1982). The size of this nick has been determined to be about 200 bp by Van Keulen, 11 11. (1985). B. Eathology 1f Sgnis1g§on1 mansoni inf11tion At the invasive stage individuals exposed to the schistosome cercariae experience dermatitis (swimmer’s itch). In the chronic phase of heavy infection most vital organs are affected. The presence of the adult parasites in the blood stream of the host is relatively harmless. However, most of the eggs produced by the female parasites are retained in various organs, eliciting vigorous host granulomatous responses (Bogliolo, I967). Granuloma formation is initiated by antigens secreted by the miracidium through pores within the rigid egg shell (Stenger 11 11., I967). Eggs deposited in the mesenteric plexus disseminate mainly into the liver and the intestinal tract, where each one evokes granuloma formation. Hith advanced pathology, collateral circulation is esta- blished; eggs reach the systemic circulation and are delivered to the 18 renal, pulmonary or central nervous system, where they initiate further granulomas (reviewed by Boros, 1989); Microcirculation studies in the livers of mice infected with schistosomiasis showed that the granulomas that form around eggs lodged in the presinusoidal capillaries impede hepatic blood flow (Bloch 11_11., I972). Symmers’ fibrosis develops in humans around the branches of portal veins after many years of infection. This fibrosis is followed by portal vein obstruction, which contributes to portal hypertension, resulting in collateral circulation. In heavy infections, continuous granuloma formation and fibrosis together with elevated portal pressure and intense imunologic activity lead to the development of hepatosplenomegaly. Hepatosplenomegaly with deranged liver function is followed by ascites fluid formation in the peritoneal cavity and esophageal varices, which rupture and cause 'fatal episodes of' esophageal and gastrointestinal bleeding. C. S ist i 1 Numerous approaches have been implemented in attempt to reduce the transmission of infection and morbidity due to schistosomiasis (WHO, 1985; Jordan, 1986; Sleigh 11_11., 1986; Klumpp & Chu, 1987). Health education, improved sanitation and water supply with focal mollusciciding and chemotherapy have been recommended as an effective means of controlling the disease (NHO, 1985). However, such strategies have suffered various limitations, and have therefore been marginally successful (Jordan 11 11., 1978; Polderman, I984; Fenwick, 1987). Vaccine development has been proposed as a cost effective means of controlling the transmission of the I9 disease. Although expectations for the development of schistosome vaccines are high (Butterworth 8 Hagan, 1987; Capron 11 11., 1987; Smithers, 1988), there is a little chance that the vaccine will be available in the foreseeable future. The complexity of host-parasite relationship, lack of knowledge of the host immune effector mechanisms against the parasite and possible evasion of the host immune system by the parasite have further tempered optimism towards schistosome vaccines (reviewed by Mitchell, 1989). At present, chemotherapy is the»most effective method for the short- term control of schistosomiasis (WHO, 1983; Liese, 1986). Since these parasites multiply outside the human.host, the worm load and oviposition are crucial for the perpetual cycle of infection. Drugs reduce both worm burden and egg excretion, thus contributing to a decline in both the intensity and prevalence of infection by reducing the population of infected snails (Cook 11 11., 1977; Pugh 8 Teesdale, 1984; Sleigh 11 11., 1986). Currently, however, few chemotherapeutic agents are available for the treatment of schistosomiasis, and the future of this approach is threatened by the possible emergence of drug resistant strains of the parasites (Bruce 11 11., I987; Coles 11 11., 1987a). Evidence for resistance to hycanthone and oxamniquine is well documented (Dias 11 11., 1982; Coles 11 11., 1987b; Yeang 11 11., 1987). The ultimate fate of the effective broad spectrum antischistosomal agent praziquantel is also uncertain. Thus, research has to be continued towards the development of new antischistosomal drugs to complement existing drugs or replace those that cease to be effective. 20 D. ‘ n ' iv Research A class of new antischistosomal compounds has been synthesized by the Hoffmann-La Roche C0., Basel, Switzerland (Stohler and Montavon, 1984). These 9-acridanone hydrazone compounds have been shown to be effective against the three principal species of schistosomes that cause human disease. The antischistosomal activity of one of these compounds, Ro 15-5458, in mice and hamsters infected with S, n1n11n1, S. ha1matobjun and S, 1121n111n is superior to other standard antischistosomal drugs such as praziquantel, oxamniquine and amoscanate (Table I). In addition, unlike praziquantel, which is active only against the invasive stages and adult parasites in 1111 (Andrews, 1981), Ro 15-5458 is effective against all the stages of the parasites (Stohler 8 Montavon, I984; Eshete, unpublished observation), a property that may be exploited for early intervention in the infection. A single oral dose of Ro 15-5458 (50, 25, or 15 mg/kg) effectively clears infected rodents and baboons of parasites and significantly reduces fecal egg excretion (Stohler 8 Montavon, 1984; Sturrock 11 11., 1985; 1987; Sulaiman 11 11., unpublished data). Limited data are available on the toxicity of Ro 15-5458 to the host. In acute toxicity studies, two different single doses were given to mice (250 and 500 mg/kg) and rats (125 and 500 mg/kg) and the number of survivors was determined 14 days after drug administration. All animals survived at the lower dose, but all died at the higher dose, suggesting the existence of a high margin of safety between the effective dose (E01, - 10.5 mg/kg) and lethal dose. In 111111 mutagenicity tests performed using S. 1ynn1nnn1nn strains TA97 and TA102 as well as repair 21 TABLE 1 Antischistosomal Activity Against S, m1n11n1, S. haen11gnjum and S. 11nnn111n in Hamsters and Mice“ Hamstersb Miceb Compound 5-1111118901 5.11111111- S. 4.19.: 1. mas 1 1.921311. Oium Ro 15-5458 11.1 8.5 41.5 10.5 Praziquantel 40.4 <50.0 74.2 272.0 Oxamniquine 126.8 inactive >400 64.1 Amoscanate 7.0 active active >100 'Reproduced from Stohler 8 Montavon, I984. "ED,o - single oral dose in mg/kg reducing the number of surviving schistosomes by 90% 22 tests with E. 1111 showed no mutagenic activity (Stohler 8 Montavon, 1984). In collaboration with Dr. R.J. Wilkins (University of Otago, New Zealand), we also tested for site specific binding and/or inhibition of DNA polymerase I activity using a nick translation system (Wilkins, 1985). At 100, 33, and 10 pg/ml of Ro 15-5458 neither binding nor polymerase inhibition was observed. These results may not be surprising in light of the absence of 1 vitro schistosomicidal action of R0 155458 (vide 1nfin1); the drug is probably converted to a biologically active product which may result in toxicity. If active metabolic products are respons- ible for schistosomicidal activity, all the tests need to be repeated. Despite extensive parasitological evaluation of R0 155458, no report is available on its mechanism of antischistosomal action. The present study was designed to examine the 1n 11111 and 1n 1111 effects of Ro 15-5458, with the intent of understanding the mode of its schisto- somicidal action. The _in vitro effects of R0 155458 on S. mansoni musculature and its relevance to the 1n yivn schistosomicidal action were examined. In addition, effects on well characterized biological processes of the parasite were studied after dosing the host with a 15 mg/kg of the drug and retrieving parasites. SUMMARY AND SIGNIFICANCE Under proper supervision, it appears that chemotherapy will play an ever increasing and crucial role in the control of schistosomiasis. A major risk in the widespread application of anthelmintics is the appearance of schistosome populations exhibiting drug resistance. This 23 necessitates the development of' other antischistosomal compounds to replace drugs that Cease to be effective. Knowledge of the biochemical/physiological processes of schistosomes is required for a "rational” design of these drugs. Unfortunately, it is still lacking for these parasites. Under such circumstances, one approach that could be used is to develop an understanding of the mode of antischistosomal action of currently available drugs and others in the process of development. Mode of action studies may provide new informa- tion on the parasite’s metabolic processes, which in turn may reveal areas for the discovery of other antischistosomal drugs. In this context, we have initiated an investigation on the mechanism of action of Ro 15-5458. It is anticipated that these studies, in addition to providing insights into the mechanism of antischistosOmal action of this novel drug, will also contribute to the limited knowledge available regarding parasite-drug interaction, opening potential areas for the control of schistosomiasis. CHAPTER II THE ABSORPTION KINETICS 0F Ro 15-5458t INTRODUCTION Knowledge of the pharmacokinetics of Ro 15-5458 is essential for its development as a potential broad spectrum antischistosomal agent, both for the meaningful interpretations of its biological effects and in the assessment of its toxicity to the host. In this section, the absorption kinetics of this drug have been described. A study of the absorption kinetics was performed after oral administration of the drug since it will probably be administered via this route. It is also important to note that oral administration of antischistosomal compounds would be most desirable for the treatment of schistosomiasis. Rabbits were used in this study due to their convenience, in model single dose pharmacokinetic studies. MATERIALS AND METHODS Ro 15-5458 [IO-2-(diethylamino)ethyl-9-acridanone(2-thiazolin-Z- yl)hydrazone] and its analogue Ro 15-9895 [lO-2-(dimethylamino)ethyl-9- acridanone(2-thiazolin-2-yl)hydrazone] (Figure 3) were kindly supplied by Dr. H.R. Stohler (Hoffmann-La Roche Co., Basel, Switzerland). 24 25 bi -~—/ :1 1 —\s / 1 H \ \ I,“ / .R CODE R Ro 15-9895 -CH2-CH2-N(CH3)2 Ro 15-5458 ~CH2-CH2-N(C2H5)2 Figure 3. R0 155458 (CaHuNfi) and Ro 159895 (CnflaNfi). 26 A. ADM Female New Zealand rabbits weighing 2-3 kg (n=5) were fasted 12 h prior to dosing but had access to water. A single oral dose of 50 mg/kg Ro 15-5458 was given as a suspension in 25% glycerol plus 1%1Cremophor EL. Five ml of blood was collected from eer veins of each rabbit before dosing and samples were similarly collected 20 min, 40 min, 1, 2, 3, 6, 12, 24, 48 and 72 h after dosing. Blood was left at room temperature to clot and plasma was separated by centrifugation at 12,000 g for 10 min. All plasma samples were frozen and assayed for R0 155458 within 48 hours of collection. B. ' - Pl The plasma level of Ro 15-5458 was determined by high performance liquid chromatography (HPLC) (Waters Associates, Milford, MA), after a simple and rapid extraction of Ro 15-5458 and the internal standard (Ro 159895) from alkalinized (150 pl '1 N NaOH) Z-ml plasma samples and plasma-based standards with 10 ml hexane-butanol (9:1) with gentle rotating and mixing for 5 minutes. Plasma-based standards were prepared by spiking 2 ml of plasma collected before dosing at concentrations of 25, 50, 125, 500 and 1000 ng/ml of Ro 15-5458 and 100 ng/ml of Ro 15-9895. Nine ml of the organic phase was then re-extracted with 250 ul of 0.01 N HCl and 100 ul of the acidic phase was injected into a NOVA-PAK Cn column (Waters Associates). The mobile phase was 0.01 M ammonium acetate (pH 5.5) plus acetonitrile (4:6) containing 0.004 M triethylamine, run isochratic at a flow rate of 1 ml/min. 27 The column effluent was monitored with a variable wavelength spectrophotometric detector set at 254 nm. Data handling and plotting were performed by a«computing integrator (Data Module, Waters Associates). The computing integrator programmed to store the standard calibration curve for each assay was used with the peak area ratio of Ro 15-5458 to internal standard to calculate the concentrations. C. U§§§_An§11§1§ Ro 15-5458, the internal standard and other prominent HPLC peaks were collected and extracted with methylene chloride. Samples were then evaporated to dryness at 37°C under nitrogen and mass spectral analysis was performed by direct probe introduction of the compounds. Electron impact spectra were determined at 70 eV on Finnigan 3200 mass spectrometer with Riber SADR data system. 0. Eh1nn111kjne1jc Analygig The half-life and the peak plasma concentration (C ) were calculated from least squares regression analysis of the elimination phase of the plasma concentration-time curve (Gibaldi 8 Perrier, 1982). The area under the plasma concentration-time curve (AUC) was calculated using the trapezoidal rule. Other kinetic parameters were calculated using the equations described for one compartment absorption model after a single dose administration (Bates 8 Carrigan, 1975; Gibaldi 8 Perrier, 1982). The goodness of fit of the experimental data to one compartment open absorption models was tested using a non-linear least squares regression analysis program (PCNONLIN) (Metzler 8 Weiner, 1985). 28 RESULTS A. ’ ati n f h mato hi Peak After HPLC separation, peaks of interest were confirmed by mass analysis (Figure 4). The retention times for the internal standard and Ro 15-5458 were 4.910.25 and 8.310.29 (mean 1 50) min, respectively. The recovery of the extraction procedure was 83_+_7% (mean 1 SD), and the sensitivity of the assay is about 10 ng. It is important to perform the extraction and injection of the sample within a short period, since the (-C=N-N=C-) bond in both R0 15- 5458 and Ro 159895 is acid labile (Figure 5). Both compounds were converted to the respective [10-2-(dialkylamino)ethyl-9-acridanone] product. Although the extent of hydrolysis was the same for both R0 15- 5458 and the internal standard, prolonged exposure of Ro 15-5458 to acid solutions resulted in a compound that interfered with the internal standard peak. 8. Eljminatjon of 30 15-S458 Plasma levels of Ro 15-5458 declined in a monoexponential fashion with time after a single oral dose of 50 mg/kg (Figure 6). This was determined from the regression analysis of the terminal phase of the plasma level-time curve (r-0.98). The same data was also fitted to a single exponential pharmacokinetic model with no time lag using the PCNONLIN program. The experimental data were well correlated (r=0.99) with the kinetic model. The elimination half-life, estimated from the average data, was 6 h. Individual elimination half-lives ranged from 3 to 7 h. 29 .mm1m-m~ a: ea ”_mapogu»= we“ sage Aces o.m . pay =c_uuage 8.1: gm ea sagauwam mmas age m. m mppcz Hmcocmuwcua-m-anum-Aocpsmpxgum_uv-~-o~a u_a:m;u=a sage m, o Foam; .=_s m 1° pm on umoumppcu =°_ou~gu 84;: cm .mmmm-m_ om Co saesumam a m_ u pagan new =_a a CO Apgv ms_u cowucmgme um umuumppou copuumem Udaz mg» seem m_ m pagan .mmcm-m_ om u_ucmgu:~ Co Ezcuumam macs as» a. < —m:~a .uuauogn mwmxpogu»; ma_ ucm mmem-m~ cm .mmwm-m~ cm 18 agaumgm mam: .e me=m_m 30 N \2 . . _ ...... 4 9:. can 2:” .5 8m own as an.“ an 2... 8m 8m 8m 2m 8m am 88 emu ELuEthlrIrrrTIp. em omm 33.2%” 3.2 as. 95 em: cm. a: am. am. 6: ma: am am on mm on a... on LIP—48E @- n E s. a? 8mm. am». so... awn 8m. 2..” EH. gum 2m 3,” 9% 8m Em 8m. new Sm on“ +2 GNN ENQGN am: cm— and GB 32 02 6mg ON. a: 90. am so am am on a? 11.—1143:. e mean.“ 1. QM Asgsumu' 3111113133 31 N\2 a. v 2:. can own sun can can a...” can own Em can 98 9mm 3m 9mm ova avm emu +2 2. 83583983:8323823328. am 8 S 8 on 3. on. jj;}~§_i1ez n Aum==.acouv v mesa.“ mu “140138 Mgsuawl 32 Nye: a; 3... 0.9”. 9.3. 02.” .02“ cum 9.”. onM. ONm 2n. can .omN. GON- 03.. OQN .onN. QvN. onN.. ... w ems—Noamom-oo.o~—3.on— 9302 ON. 0366- am 00 ON 3 an 0.. on ._\ .M , .ahn. 0.2". Non. own can ONJ 0.3. 9.6” oaN. JON. QNN. OWNP 0.0m OWN. JnN. JNN 2N uppn- .— E .2“ 0802 8- 9N. on. on. a: an. em. a: can an on Ow on a? on {14 . .mu .8 a. .eose..=ou. v o.:a.. 33 9.19 I —. TM 06 l Jul/i ;QLJ C Figure 5. Chromatograms of Ro 15-5458 and Ro 15-9895. Chromatogram A is an extract from plasma of a rabbit. Peaks at retention times 5.06 and 8.53 min are from internal standard and Ro 15-5458, respectively. Hhile peaks at retention times 2.6 and 3.86 are for the [lO-Z-(dialkylamino)- ethyl-Q-acridanone] products of Ro 15-9895 and Ro 15-5458, respectively. Chromatogran B is for Ro 15-9895 and C is for Ro 15-5458 after exposure of both compounds at room temperature to 0.01 N HCl for 30 days. 34 ..mm.ou.. :c.mmm.am. mousse. .mao. cao=._-=e= a: co.... was ac.— agp ..m.=. mace —aco ax\ms-cm a a=_=a_—c. mama—g e. =c..~..:ou=ou mmem-m. a: om~go>< ? V m3: .o a.:m.. (tux/BU) BS‘pQ—SL ca ;,0 uogoquaouoa 501 35 C. Apsgrptign of R0 15-545§ Ro 15-5458 appeared in the general circulation abruptly and declined without a plateau (Figure 7; Appendix I). An average peak plasma concentration of 700 ng/ml was observed 2 h after dosing. However, results from individual rabbits suggested variable bioavailability of Ro 15-5458 after oral dosing. Maximum plasma levels ranged from 500 to 1200 ng/ml (1.3-3.1 uM). Visual inspection of the plasma level-time pattern for individual animals suggested that the data were consistent with zero order absorption (Figure 8). To confirm this hypothesis, we fitted the average data to a one-compartment model, with either first-order or zero-order absorption, using the PCNONLIN program. Based on the correlation coefficients, the estimated pharmacokinetic parameters and their standard error of'mean, the goodness of fit of the experimental data was better for the zero-order absorption kinetic model (Appendix II). Therefore, this model was used to calculate apparent kinetic parameters (Table 2) for the oral absorption of Ro 15-5458. The apparent rate of absorption (kw/V), the lag time (to) and the duration of absorption (T) have been well estimated with this absorption model. DISCUSSION The absorption of drugs from the gastrointestinal tract after oral administration is a complex process. It is easier to study pharmaco- kinetics after intravenous infusion, as more reliable kinetic information 36 . .. x.u=maa< :— =zogm m. “Pans; —~=u_>_uc_ soc. ..ammm .Amuzv m._nn~. a. omov u—a:_m mx\ma-cm a ma =o_uagum.=.sua _agc as. m=.3o.—cu me.» .o =o_.u::. a ma mmvm-m. om mo m=c_aasacwu=ou «Ema—m .s og:m.. 3 .2: N,“ I n’* h I+IN D INI’ P m P w h m I C I‘ll IIII'G I, II nu . oII / . 8N '/ I 11.11 / / § § (IN/5°) sets-9t on 1° "emu-om nus-ma 37 .55.... 2.2. 3.2.... 539.3% ..oEo-Em~ a 9...: 3:; 3.2.6.3 .2... 3.3 E; 3.52. 2.52235 . O 2.3. .32. 2.8 29:... 3}... cm a .3... 3.5 3:39. 5 Semé. 3. 2. 3:9... 2.3: .o 9:5: 3 we: E: in * ( (Lu/Bu ) 99:79—91, 03 I0 uogmauaouoo owsold 38 TABLE 2 Pharmacokinetic Data from Rabbits Following a SO-mg/kg Single Oral Dose of Ro 15-5458 Unless otherwise stated, parameters were calculated from the average plasma level (n-5). Apparent elimination rate constant (k,), h“1 ~ 0.12 Maximum plasma level (Emu), ug/ml 0.7 Apparent rate of absorption (kw/V), mg/h-L‘ . 0.53 Area under plasma level time curve, (AUC) mg-h/L 5.8 Apparent onset of absorption (to), minb 6.0 Apparent duration of absorption (T), h° 1.5 'Average value calculated using plasma level and the corresponding time from each rabbit. The following equation was used: cm - kJVk, l-e'K-T bCalculated using the following equation: c - ko/Vk, 1-e'K-(t’to’ and plasma level and corresponding time (t) of the distribution phase, i.e. to is the mean lag time, calculated using each data point obtained during absorption (T). °Calculated using the following equation: AUC - kol/Vk, 39 can be obtained from the plasma level-time data. In the absence of such data, however, the plasma level-time data obtained after oral administra- tion are also useful. It is particularly important to obtain such data if it is intended to administer the drug by the oral route. The absorption of Ro 15-5458 from the gastrointestinal tract of rabbits was very rapid, the maximum concentration of the drug in the peripheral blood being reached within 1 to 2 h. However, Ro 15-5458 showed variability in the extent of appearance in the blood, as determined by the AUC and Cmam values from the individual rabbits. The source of this variation is not clear. However, it is possible to make certain speculations based on some preliminary observations made in our laboratory. The presence of food in the gut of the rabbits significantly reduced the extent of absorption of Ro 15-5458. Therefore, the rate or extent of drug appearance in the blood could vary depending on the residual food in each rabbit after 12 hr of fasting. In this regard, it is important to note that food was observed in the stomach of some animals after I2 h of fasting. It was not possible to fast rabbits for longer‘ periods, since toxic effects, including convulsions, were observed. Another potential explanation for the observed variability could be inter-animal differences in excretion or metabolism. Urine samples collected from two rabbits between 0 and 2 h after dosing were analyzed by HPLC. During this period, about 6% and 11% of the measured maximum plasma level of the drug was excreted unmetabolized by these animals. Variations may thus arise from differences in the excretion of the drug during the time of absorption. 40 These observations suggest the need for modification of the dosage form before any therapeutic or toxicological evaluation of the drug can be performed in other species. It may also be necessary to examine the time interval between food intake and drug administration, since the amount of food in the gastrointestinal tract may be a factor in either the toxicity or the efficacy of the drug. Schistosomes live as adults in the blood vessels of the host. Rational treatment of infection with these parasites requires maintenance of an optimal level of the chemotherapeutic agent in the general circulation, so that a balance is maintained between toxicity to the host and efficacy towards the parasite. Thus, understanding the plasma kinetics of Ro 15-5458 is important for its development as a potential broad spectrum antischistosomal agent. Extrapolation of the rabbit data to other definitive hosts of the parasite may not be straightforward. However, the overall absorption and plasma pattern of the drug in rabbits furnishes important preliminary information that will be required before human trials. The rabbit data are particularly interesting in terms of the toxicity of Ro 15-5458. A preliminary acute toxicity study reported previously by Stohler and Montavon (1984) described the death of all mice 14 days after treatment with a single oral dose of 500 mg/kg. 0n the other hand, a single oral dose of 15 mg/kg completely clears mice of worm burden (Eshete, this thesis). It is possible to expect low plasma levels of the drug in mice at this low dose, perhaps indicating a sufficient safety margin between the therapeutic dose and the toxic dose. In treatment 'of plasma level-time data obtained after oral administration of drugs, the absorption kinetics have been invariably 41 assumed to be first-order. This notion has been challenged by evidence that, at least under certain conditions, the gastrointestinal absorption of several drugs, including ethanol (Cooke, 1970), sulfisoxazole (Kaplan gt 31., 1972), erythromycin (Colburn & Gibaldi, 1977), hydroflumethiazide (McNamara, Colburn & Gibaldi, 1978) and griseofulvin (Bates 8 Carrigan, 1975), is best described as an apparent zero-order (constant rate) rather than first~order process. The plasma level-time profile of orally administered Ro 15-5458 in rabbits also appears to follow a one-compart- ment pharmacokinetic model with zero-order absorption, first-order elimination. The lag time was short, which suggests an abrupt appearance of the drug in the circulation. Drug levels peak and decline without a plateau, consistent with zero-order absorption. SUMMARY The absorption kinetics of Ro 15-5458, a new antischistosomal drug, was studied in rabbits following the administration of a single 50 mg/kg oral dose as an aqueous suspension in 25% glycerol-1% cremophor EL. Ro 15-5458 was absorbed from the gastrointestinal tract rapidly with a lag time of about 6 min and declined without a plateau with a half-life of about 6 h. Maximum plasma levels ranged from 500 to 1200 ng/ml (1.3-3.1 pH). The plasma concentration-time profile of Ro 15-5458 after a single oral dose appears to follow a one-compartment pharmacokinetic model with zero-order absorption, first-order elimination. CHAPTER III THE lfl VITRQ EFFECTS OF R0 15-5458 GM ADULT §CflISTOSQMA MANSONI INTRODUCTION Three drugs have been recommended for the large-scale chemotherapy of schistosomiasis: metrifonate for S. haematgbigm, oxamniquine for S. manggni, and praziquantel, which is effective against all the species of schistosomes responsible for the human disease. Metrifonate is inexpen- sive but requires several doses, and this makes its delivery expensive (Korte g1.al., 1986). Oxamniquine is not well tolerated by patients, and strains of parasites resistant to the drug have been reported in Brazil (Dias g1 a1., 1982) and Kenya (Coles gt al., 1987b). One other drawback with drugs, which are only effective against a single species of the parasite, is that multidrug therapy may be required in areas where more than one species is endemic, which is undesirable for medical and economic reasons. Praziquantel has a broad spectrum of activity against these parasites. Single oral dose administered for patients in most areas is well tolerated, but the cost of praziquantel continues to limit its use in developing countries. The possibility of appearance of drug-tolerant or resistant parasites must also be considered when treatment reaches a large sector of the infected population. 42 43 Another broad spectrum antischistosomal agent that is in the process of development is Ro 15-5458. The antischistosomal activity of this com- pound has been demonstrated to be superior, in experimental animals, to praziquantel and other standard antischistosomal drugs (Stohler 8 Montavon, 1984). Preliminary work from our laboratory indicated that administration of a 15 mg/kg single oral dose of Ro 15-5458 to mice infected with S. mansggl effectively clears the infection. The drug was effective when administered i.p as well as i.m (Eshete, unpublished data). However, the in 21339 incubation of adult schistosomes with reasonable concentrations of the drug did not result in parasite killing. In the meantime we observed significant stimulation of the parasite musculature after exposure to Ro 15-5458 in yitrg. Since the maintenance of the integrity of the neuromuscular system of schistosomes in 1139 is critical for reproduction and perhaps in the feeding behavior of the parasites, in this section an attempt was made to characterize the effects of the drug on the musculature of schistosomes and to determine the relevance of this in yitgg effect to the in 2119 schistosomicidal action. Attempts were also made to generate active metabolites of the drug since the parent compound has no l__ yitro activity. Drug effects on some vital physiological processes of the parasite were also examined. 44 MATERIALS AND METHODS A. t' a 't c v A Puerto Rican strain of S. mansgni maintained in laboratory-reared Bigmnhglgriapglghrgta,and outbred female white mice was used in the study. The method of infection was by i.p injection of 200-300 schistosome cercariae. Adult parasites were isolated from infected mice 6 to 7 weeks . post-infection as previously described (Bennett and Seed, 1977). Briefly, mice were killed with cervical dislocation and were dissected for parasite removal. The parasites collected from the mesenteric and intrahepatic veins were then placed in RPMI-1640 medium (Gibco, Grand Island, NY) buffered with 25 mM Hepes [4-N(-2-hydroxy ethyl)-1-piperazine ethane sulfonic acid] (Sigma Chemical Co., St. Louis, MD) to pH 7.4. Parasites from at least 5 mice were pooled and randomized into groups before any experimental manipulation. In experiments where single parasites were used, males and females were separated using a dissecting microscope and fine forceps after exposure in RPMI-l640 to 0.5 mg/ml sodium pentobarbi- tal. B. In yitrg Parasite Cglturg Long-term parasite cultures were performed in Eagle’s minimum essential medium (Dulbecco modified) supplemented with 20% calf serum containing 8 ug/ml of gentamicin and 1 ug/ml of amphotericin B at 37°C in 5% CO,/95% air. Duplicate dishes containing 7 parasites in 3 ml medium were incubated with Ro 15-5458 at a final concentration of 1 or 10 pM. Control parasites were incubated in parallel with treated parasites. One group was incubated for 3 h and the other for 3 days. After a single 45 application of the drug and incubation for either 3 h or 3 days, the medium was replaced and incubation was continued for 20 days, changing the medium 3 times a week. Each parasite was viewed under a light microscope for contractile activity in a 10 pH 5-HT containing medium. The absence of response to the 5-HT was used as an index of parasite death. C. W These experiments were performed using the method previously described by Cioli (1976). Twenty male parasites were exposed to 10 DM of Ro 15-5458 in RPMI-1640 medium buffered with 25 mM Hepes, pH 7.4 containing 20%. calf serum. Parasites were washed and surgically transferred to the mesenteric vein of uninfected Nile rats (Arventicus Ms) either after 3 h or 3 days of in D119 drug exposure. Parasites were retrieved from the recipient animals by perfusion 2 weeks following transfer and the number of viable parasites was determined. D. M ic A vi R ordin - Contractile activity of paired parasites was measured as described by Bennett 8 Pax (I987). Parasites were randomized into 10x75 mm thin wall glass tubes (3 pairs/tube) containing 1.3 ml of Hepes-buffered RPMI- 1640 medium and were preincubated for 15 min at 37‘C. Stock solutions of all 9-acridanone hydrazone compounds were made in dimethyl sulfoxide (DMSO). Desired final concentrations of the drugs were achieved by transferring aliquots of the stock and the same volume of DMSO to treatment and control tubes, respectively. Incubations were continued for 30 min and the contractile activity was measured for 20 seconds with a 46 motility meter. Lactic acid analysis was also carried out on media aliquots by monitoring the rate of reduction of NAD’ at 340 nm during the conversion of lactate to pyruvate by the enzyme lactate dehydrogenase (Schon, 1965). Longitudinal muscle tone of'male parasites was recorded as described by Fetterer g1 31. (1977, 1978). Briefly, parasites were placed in a recording chamber containing 2.5 ml of RPMI-164O medium at 37°C. After the hook-up of parasites to the system, a 5 min equilibration period was allowed before the application of any treatment. Aliquots of 10 mM stock solutions of the 9-acridanone hydrazone compounds in DMSO were added to the recording chamber to desired concentrations such that the amount of DMSO did not exceed 1%. Tension was monitored by means of a non-flexible suction pipet made of polyethylene tubing (id 0.38 mm, od, 1.0 mm) attached to the tail end of the worm by mild suction applied with a syringe. The second pipet (flexible) with the same internal and external diameter was then attached 0.75 to 2.5 mm anterior to the non-flexible pipet. The attachment of the wire to the flexible pipet was such that any movement of the pipet produced an up or down movement of the flag. The flag was oriented such that it interrupted the light path between a light source and a photodiode whose output was attached to a chart recorder by way of a DC amplifier. With this arrangement, any contraction or relaxation of the muscle in the worm resulted in a change in the light reaching the photodiode and was consequently recorded as a voltage change by the chart recorder (Figure 9). .daqwaua .w o—as ..zua c. co.m:oa «puma: —a=.c=..m=op as. scoumc a. can: 8:.acaaaa mg. .8 =o..a.:mmccamc u..a§mgum .a c.=a.. 47 3243.3... / 31.3.3 _ _ Oh - 5.2.. I, giggcgéee XNIII. # .31.: / 43.303 30:33.... 48 Since the force developed by the parasite depends on the distance between the two pipets, the tension developed is expressed in milligrams per millimeter of parasite length. Data presented in tables and elsewhere in the text, are the change in tension 10 min after addition of the agent compared to the tension just prior to its introduction or, for controls, after addition of the vehicle (DMSO). In cases where two or more pharmacological agents were introduced sequentially, data presented represents the difference in tension between the second and the first drug recorded for 10 min in both cases. E. Egg Erodggtign Aasay The in vitro effect of Ro 15-5458 on the fecundity of §. manaonj was determined as described by Morrison a; a1. (1986). Briefly, 15 paired parasites were placed into sterile Erlenmeyer flasks containing 50 ml of RPMI-164O medium described above containing 50% horse serum plus 100 U/ml penicillin, 100 ug/ml streptomycin (Gibco, Long Island, NY) and 50 pH 3- mercaptoethanol. Ro 15-5458 was dissolved in DMSO and was added in a volume not exceeding 50 pl. Control flasks received the same volume of drug-free DMSO. Parasites were then incubated at 37°C in an oscillating water bath for 72 h. After incubation, each flask was shaken and three 5 ml media aliquots were placed in a gridded petri plates and eggs counted under a dissecting microscope. F. n r r o r ors Parasites recovered from the same infection date were randomized into tubes (6 males/tube) containing 1 ml of RPMI-164O medium. Parasites 49 were preincubated in a water bath at'37°C for 30 min and then Ro 15-5458 at the final concentration of 10 MM was added to treatment tubes. Both control tubes (containing 0.1% DMSO) and drug treated tubes then received 10 uCi/ml of either L-[4,5-’H]leucine (50 Ci/mole), [methyl -"H]thymidine (74 Ci/nlnole) or [5,6-3H]uridine (45 (ii/mole) (all from ICN Radiochemicals Irvine, CA). After a l h incubation, parasites were washed 3X with ice cold saline, homogenized and then sonicated in 1 ml of dHZO. The homogenate was divided into two equal portions and both were treated with 5% trichloroacetic acid (TCA). The precipitate in one of the fractions was collected on glass fiber filters (Hhatman GF/C) using a vacuum- operated filtration apparatus (Millipore Corp., Bedford, MA). The filters representing the TCA-insoluble portion, were transferred to a glass scintillation vial containing 250 pl of NCS tissue solubilizer (Amersham, Arlington Heights, IL) and 100 pl of glacial acetic acid and were incubated at 60°C for 20 min. After solubilization, the vials were brought to room temperature, 10 ml of ACS aqueous scintillant was added, and radioactivity was measured with a Beckman LS7600 scintillation spectro- meter. The other fraction was centrifuged at 10,000 g for 10 min and the resulting supernatant was used to measure TCA-soluble radioactivity. The pellet was dissolved in l M NaOH for protein determination with the method of Albro (1975) using bovine serum albumin as standard. 6. Inggbatjgg 9f Parasites in Drug-Metaboliaing System. 1. S r f v Infected and uninfected ICR/BR Swiss Webster female mice (Harlan Sprague-Dawley, Indianapolis, IN) were treated either with sodium 50 phenobarbital (3x80 mg/kg, i.p) for 3 days or Ro 15-5458 (2x15 mg/kg, p.o) at 8 h interval. Mice from both treatment groups were killed 24 h after the last dose by cervical dislocation and the livers excised and placed in a sterile, ice-cold beaker. 2. ' n f i n A liver homogenate fraction enriched in microsomal drug metabolizing enzymes was prepared as previously described (Ames at al, 1973). All the apparatus and solutions used were sterile and precooled to 4°C; the temperature of the tissue was kept below this temperature during the preparation. The livers were washed in 0.15 M KCl, blotted, minced with scissors in the same solution (3 ml/g of wet liver) and homogenized in a glass homogenizer with a Teflon pestle driven by a Con- Torque power unit (Eberbach Corp., Ann Arbor, MI). The homogenate was centrifuged for 10 min at 9000 x g and the supernatant ($9) decanted and saved. One ml aliquots of the supernatant (microsomes from 0.4 g of wet liver) were quickly frozen in dry ice and stored at -80H:. 0n the same day, a portion of the supernatant was tested for activity by measuring aryl hydrocarbon hydroxylase (Van Canfort at al., 1977) and benzphetamine- N-demethylase (Prough and Ziegler, 1977) activity. 3. Praparatjgn 9f NADPH-Reganerating System Three different systems were used for drug activation; the standard 0.1 M phosphate, pH 7.5; RPMI-l640 buffered with 20 mM Hepes, pH 7.5; and Krebs-Ringers tris maleate (KRTM), pH 7.5. The composition of KRTM is 120 mM NaCl, 4.8 mM KCl, 2.6 mM CaCl,, 1.2 mM MgSO, and 5 mM glucose (Read at a1., 1963). All the systems contained 340 M NADP’, 81 51 pH NADPH, 300 uM NADH, 4.8 mN glucose-6-phosphate and 2 U/ml of glucose- 6-phosphate dehydrogenase. The phosphate system also contained 6 mM MgCl,. 4. MW Ten pairs of parasites (n-3), were placed in a dish containing 3 ml of filter sterilized RPMI-164O medium, 25 uM Ro 15-5458 and 1 ml of S, from uninfected or infected phenobarbital-treated mice. The dishes were then incubated at 37°C for 1 h. Simultaneously, 2 ml of phosphate system with 1 ml of S, and 100 M Ro 15-5458 was incubated at the same temperature for 2 h without parasites. One ml of this mixture was added to another group of dishes containing 10 pairs of parasites in 7 ml of RPMI-1640 medium (14 mM phosphate and 12.5 uM of the original drug) and incubated for 1 1L. Similar experiments were performed with S, from infected and uninfected Ro 15-5458 treated mice. Other dishes with only 5,, 25 pH Ro 15-5458 or 1 ml of S, and 1% DMSO (the vehicle used to dissolve drug) were also simultaneously incubated. *After 1 h of preincubation parasites from all dishes were washed in RPMI-1640 and were incubated in 10 ml of RPMI- 1640 medium containing 20% calf serum, 8 ug/ml of gentamicin and 1 ug/ml of amphotericin 8 up to 15 days changing the medium 3-times a week. I. 1 _p. '91 . '. . ' - i. P . 1. '- ov‘ ec from Tre. e- 'a-nit_ A 50 mg/kg single oral dose “of Ro 15-5458 was administered to rabbits (n-2) and plasma was recovered 6 h after dosing by cardiac puncture. The plasma was divided into two equal volumes and one of the portions was heated at 55°C for 1 h to inactivate complement, while the other fraction was used for parasite incubations without further treatment. Fifteen pairs of parasites were incubated in RPMI-1640 medium 52 containing 50% plasma from dosed rabbits, 8 ug/ml gentamicin and 1 ug/ml of amphotericin B. Incubation was continued for 10 days after replacement of the rabbit serum with 10%.calf serum in RPMI-1640 medium, changing the medium twice a week. RESULTS The effect of different concentrations of the drug on the motor activity of paired parasites is shown in Figure 10. The stimulatory effect of 100 pH of the drug was exhibited for a short time (ca 1 h) and declined dramatically within the next 10 h. With 1 uM of Ro 15-5458, a marginal stimulatory effect was observed, while 10 M significantly stimulated the parasites, an effect which occurred quickly and lasted up to 48 h. Metabolic requirements of the parasites were also raised after incubation with Ro 15-5458; the amount of lactic acid produced by the parasites was correlated with the degree of muscular contraction (Table 3). The stimulatory effect of 10 pH Ro 15-5458 was reversed by the cholinomimetics, carbachol (100 uM), physostigmine (10 pM) and arecoline (1 uM) (Table 4). Longitudinal muscle tension of adult male 5. maflsgn_i was also recorded in the presence and absence of Ro 15-5458 in the medium. R0 15— 5458 caused a dose-dependent increase in the muscle tone (Figure 11); drug concentrations greater than 50 pH cause acute toxicity to parasites, thus reducing the muscle response observed at these concentrations. A similar study'was undertaken to compare Ro 15-5458 with other compounds which have been previously reported to have effects on schistosome longitudinal 53 4...... a a cum: 8:. mamomosamc .=.cq «use gum. .mczmcaxo ca..a um.au.u=. «83.. an uugamaoa Aa.—.aaa use 2:. co. can a. _ mo =c..a..=ou=au —a=.. mg. as ave.-_z¢¢ c. mm._m~.~a m:.=_a.:ou moss. a. emuauosac. ma: use: .muu.a.=q.flmqwmma .w ca..~a .o a..>..ua m...u~..=cu co mmcm-m. oz .8 gum... .o. oc:m.u A5 2.... .3333... 3 8..” 4w. m..o...m.m..v.m.o ov a" ... m .03 m. 8. W ecu mm .aou .oou .mw .oun 54 TABLE 3 In vitro Effect of Ro 15-5458 on Lactic Acid Production by S. mansoni Incubation Control 4 Ro 15-5458 (uM) Time (h) 0.1% DMSO 10 l 24 2.410.7 ° 7:1.0 Z.7i0.3 3.3:0.1 48 5.7iO.7 11:2.0 6.3i0.3 6.7:0.7 Values are lactic acid excreted to medium, mean 1 SD, in umoles/ pair (n-6). 5.5 TABLE 4 Effect of Ro 15-5458 on the Contractile Activity of Adult Paired S. mansanj and Reversal by Cholinergic Agonists Motility Index Treatment (HM) (arbitrary units) Control (0.1% DMSO) 156:35 Ro 15-5458 (10) 242149° + Carbachol (100) 25: 5b Carbachol (100) ' 24:10 + Ro 15—5458 (10) 81124 Control (0.1% DMSO) ' 115:14 Ro 15-5458 (10) 295:48 + Carbachol (10) 211163 Carbachol (10) 36: 7 + Ro 15-5458 (10) 238¢31 Physostigmine (1) 37:13 + Ro 15-5458 (10) 174:53 Motility of control parasites and parasites exposed to the first drug‘ was recorded. The second drug was then introduced and the motility measured 30 min after the second drug’s introduction. Results are mean i SD (n-5). 56 macmmmcgmc .=_oa mama seam .mmcagu «as. an :mxa. ma: AEE\ms ~.m. m=—~> 8.;p .m> =o.u~g.:mu:ou .o .c—a _aucca.umg m—azou as. 28.. eu=_a.m.mv ma: m=_~> 5:5.xas as» .w opaa upsvm .o cc.m:m. w—umzs use mmvm-m. oz cmmzuwn 8.;m=o_uapmc mmgcnmos-amoa 923 mm¢mlm— cm .0 :ozobcoocoo 90.. o «I N p ......5 8 m =3... 2: .co.m:wa a. mmcagu .dqmwmma ... 38:... \\\\\i 11...)... CD -ON -O¢ .00 .00 roo— (wnngow )0 x) uogsuaa apsnw u! abuouo 57 muscle tension (Mellin at al., 1983, Pax at al., 1984). The effects of atropine and 5-HT were (both 20 uM) compared to the same concentration of Ro 15-5458; Ro 15-5458 showed a more pronounced effect on longitudinal muscle tension than either atropine or S-HT (Figure 12). The ionotropic effect of Ro 15-5458 was reversed by washing and was antagonized by 100 uM carbachol, 10 uM physostigmine, 1 uM arecoline and 100 uM of the 5—HT antagonist metergoline; physostigmine was more potent than carbachol in its action (Table 5). Carbachol causes relaxation and flaccid paralysis of schistosome longitudinal muscle. Ro 15-5458 altered the magnitude of the response to carbachol when it is simultaneously introduced in the recording chamber i.e., the dose of carbachol required to elicit a particular magnitude of response is increased by Ro 15-5458 (Figure 13). The effect of other structural analogs of Ro 15-5458 on the musculature of male schistosomes was also studied using the same technique. A stimulatory effect on the parasite musculature was observed with all the compounds containing an aminoalkyl side chain at the 10 position of the acridine ring. Substitution of this functional group with -CH, (Ro 14-7918), -CH,-CH,-CH,-CH, (Ro 15-3149) or -CH,-CH,-0-CH, (R0 15- 3455) diminished the muscular effects (Table 6). The effect of the drug on in vitro egg production as well as on the uptake and incorporation of DNA, RNA and proteinprecursors into the parasite'macromolecules was also examined. The results of the egg production experiments (Table 7) showed no difference in egg output between parasites treated with 10 uM Ro 15-5458 and control parasites. 58 Figure 12. The effect of 20 11M Ro 15-5458, atropine and 5-HT on the longitudinal muscle tone of adult male S. mansaai. Drug was introduced at the arrow after 5-10 min of equilibration. ATR - atropine; 5-HT - 5- hydroxytryptamine; R0 - Ro 15-5458. 59 TABLE 5 ' Effect of Ro 15-5458 on the Longitudinal Muscle Tone of Adult Male S. mansgnj and Reversal by Cholinergic Agonists Change in Muscle Tone Treatment (uM) (mg/mm) Ro 15-5458 (10) 2.5:1.2‘ + Carbachol (10) 0.6:0.7° Ro 15-5458 (10) 2.1i0.8 + Carbachol (100) '4-7i2-3. Ro 15-5458 (10) 3.9il.7 + Physostigmine (10) '0-7i0-3 Ro 15-5458 (100) 3.1il.8 + Arecoline (l) -6.7¢3.0 Ro 15-5458 (10) 3.3il.8 + Metergoline (100) -1.2i0.6 Carbachol (10) -1.6¢0.l + Ro 15-5458 (10) 2.4iO.9 Carbachol (100) -3.1¢1.0 + Ro 15-5458 (10) 2.0¢O.7 Physostigmine (10) -2.5¢l.3 4 + Ro 15-5458 (10) -0.9i0. After recording the basal muscle tone, two drugs were introduced sequentially. ‘Change in tension 10 min after introduction of the first drug as compared to the tension prior to treatment. I’Change in tension 10 min after introduction of the second drug as compared to the tension just prior to the second drug’s introduction. Results are the mean i SD (n=5). 60 ..mn:. cm H came as. mucmmocaa. u=.oa «use scam u mmccama. o>.ua—og .co.m=m. m—umza puma; «a. m=_c.cumg sm..~ umssmams «a: mmem-m_ a: mz—a —oguaa.au cc poguangau o. magaamug asp ..ogunngau o. co.m=uu «Fonz: pa=.v=..m=o_ macmoam.=um mo mmgcama. mg. cc omvm-m_ oz .8 uumumm asp .m. wean—m $43 3:02:00 .0 cozozcoocoo 00.. n N — 0 .1 NI . A. oz :3 a... 3:02.504 oz 2: 2.0 + .ozocncooo . _ozooncooo. . 0.. W a. ' om. .- J . 8.“ a a. a. r nu 3 .- ..ugn nfié. 61 .8 Boom 3-225%: 8.: .3 35.53% 2.: 88. 63:. 8.8: 352.28 252.62 m:c=mc€u~-m 9: we :1 S E; 8.3 ......o 5 33:83 .3938: .... 25:83.. ma um=.scmpmu ma: www.mmcmq nmc_ma mo x._..uumcu=ou van wpas mo cc.mcmu 8.8mae .mc.u=u.m=o.. ..ccmcms .m apaum ma mu.p.uuacucou use mcoa 8.8m:2 .mcwvauwmco_ mg» co mmopmcm mu. new mmem-m. am we mpummmu .o mpnmh ocmxma m.o_ m.o_ ca_A c¢_A o¢_z --- ounce «_mg.m 3<§ 8... 5 as °m_«~om o~_«mmm ¢~_Hcmm mvwme_ -H_~_ mmfl~m_ _mwmq~ .xmu=_ »u___ucz ~.ouc.n q.ofi ~._ m~.cwe._ m.cfim..c m.cm~o.c ..cmmo.c --- z: oo. _.¢u~._ m~.c«~m.o ~.efi_._ m_.cfio_.¢ ~_.c+m~.c ~.o+~m.c --- z: a. «gag mpumzs :. macagu “who-_~ a: mama-m_ oz mmcm-m_ ca mm.m-m_ a; a¢_m-m_ a: a_m~-¢_ a: _agagou o ua=

_uu~o_vmc open—om=_- ma mmqm-m_ am a» camcaxm qumqma .m apau< cu=_ «Lonesome; m>wuuaomvam mo co_umcoqgcu=_ tea mxmaa: m uamATPase (Levin &, Neiss, 1977, 1979; Roufogalis, 1982). Inhibition of the schistosome calmodulin-dependent bovine heart adenosine 3’,5’-cyclic monophosphate phosphodiesterase by these compounds (Thompson 91 11., 1986) has also been demonstrated. In addition, these investi- gators observed acute inotropic effects on the longitudinal musculature of parasites after exposure to the phenothiazines. The existence of Ca“-Mg”-ATPase in whole worm homogenates and tegument fractions of S. mansoni has been reported by several investi- gators (Nechay gt 31., 1980; Cesari g; 31., 1981; Podesta & McDiarmid, 1982). The schistosome enzyme is inhibited by phenothiazines but is activated by cholinomimetic agents (Semeyn, 1987) and exogenous calmodulin (Cesari g1 al., 1981). The parasite enzyme is presumed to function like the mammalian enzyme in the extrusion of ca” from the cytosol. It is thought that the manner'by'which cholinergic agents exert their inhibitory action on schistosome mechanical activity is through the activation of a ca”-Mg”>ATPase, which brings about a net efflux of ca” from the parasites (Semeyn, 1987). Phenothiazines by inhibiting the enzyme, increase the internal [Ca“] and increase tone. Ro 15-5458»may act in a similar fashion on the parasite musculature, i.e., it may inhibit the Ca”-Mg”-ATPase, an enzyme presumed to be important for the actions of cholinomimetic agents. This inhibition would increase intracellular [ca”] and promote contrac- tion. Antischistosomal compounds, such as praziquantel , the benzodiazepine derivative, Ro 11-3128 (Pax,g1flal., 1978) and alkyldibenzylamines (Bueding 70 & Penedo, 1957), cause paralysis of the parasite musculature. The resultant immobilization of the parasite suckers results in the dis- lodgment of parasites by the host blood flow (Andrews, 1981; Mehlhorn gt a1., 1981). Drugs that affect parasite musculature are thought to cause slow death by depriving parasites of nutrition. However, the muscular effect of Ro 15-5458 does not appear to be related to its schistosomicidal action. Evidence for this observation comes from the following: (1) The inactive analog, Ro 21-6787 (ED“,> 5 x 300 mg/kg), is as potent as Ro 15-5458 (EDm, 10.5 mg/kg) in increasing the muscle tone and motor activity of the parasites. (2) The concentration-response curve of Ro 15-5458 on parasite musculature suggested that the threshold concentration, required to produce significant changes, is above 5 uM. It is doubtful that this concentration can be achieved after dosing mice with a 15 mg/kg, Judging from the absorption kinetics data in rabbits (Eshete & Bennett, 1990). (3) Absence of schistosomicidal activity upon long-term l. yitrg incubation and the absence of a hepatic shift even 4 days after dosing also suggest that other mechanism(s), unrelated to the muscular effect, must be responsible. Nevertheless, a report on the. E090 of the 9-acridanone hydrazone compounds (Stohler & Montavon, 1984; Stohler, personal communication), together with the 1n_y1trg experiments on the parasite musculature (Table 6) suggest the existence of a relationship between the schistosomicidal activity and presence of the aminoalkyl side chain. Low antischistosomal activity was observed when this group was substituted with hydrogen or a 71 methyl group (Sturrock gt 11., 1987). The reason for this observation is not known at this time. However, it appears that the thiazolinyl group at position 9 of the acridine ring is vital for the schistosomicidal action of these compounds, although the relative potency may depend on the presence of an aminoalkyl side chain, perhaps due to pharmacokinetic reasons. Studies on vervet monkeys in Sudan (Sulaiman gt gl., unpublished data) and on baboons (Sturrock gt g1., 1985; 1987) demonstrated the efficacy of Ro 15-5458 in reducing both the output and viability of eggs. Prompted by these results, the effect of this compound on the incorpora- tion of precursors into parasite macromolecules was examined, since the synthesis of these macromolecules has a direct bearing on egg production and hatching ability. ‘The presence of Ro 15-5458 in the incubation medium has no influence on either the uptake or incorporation rate of the precursors; this is in contrast to parasites treated in yiyg, where the opposite result was observed. The results of the 13,11t1g incubation and parasite transfer experiments suggest the absence of it yittg schisto- somicidal activity of Ro 15-5458. Why the drug needs the host system to exert its lethal action is not clear. It may be converted to an active metabolite(s), although this could not be simulated in the in yitrg experiments. It is also possible that drug may be converted to an active form by stomach acid, since we noted in yttng schistosomicidal effect with Ro 15-5458 hydrolyzed with 0.01 N HCl and pH adjusted to 7.4. Another speculation is that the drug could bind to the host erythrocytes, ingestion of which by the parasites in sufficient amount would cause toxicity. This possibility was examined 72 in yitrg, but the‘parasites did not consume cells in culture. Attempts to induce the cytochrome P450 enzymes by prior administration of sodium phenobarbital or inhibit them by the administration of SKF 525A also did not affect the activity of the drug. Parasite elimination in 31le may also require additional host- dependent immunological events. However, in light of the absence of any structural or functional damage to the parasite surface membrane (Chapter IV) after dosing with Ro 15-5458, there is skepticism about the contribu- tion of the cellular imunological response of the host to parasite killing at the initial phase of parasite drug interaction. SUNHARY No schistosomicidal effect could be demonstrated when pairs of S. mgnsgni isolated from mice were transferred into a medium containing a solution of Ro 15-5458 (10 pH) for 72 h. However, the drug caused a significant stimulation of the parasitermusculature at this concentration, increasing both the contractile activity and longitudinal muscle tension. This property of the drug appears to depend on the presence of the aminoalkyl functional group at the position 10 of the acridine ring, since analogs lacking this group did not cause similar effects. An attempt was made to characterize the drug effects on the parasite musculature and determine the contribution of this effect to the in yiyg schistosomicidal action of the compound. It appears that the drug actions on the parasite musculature are dependent on processes normally modulated by the cholinergic system, since concentration-dependent mutual antagonism was observed between Ro 15-5458 and cholinomimetic agents. Although drugs 73 that affect parasite musculature are known to cause slow death of parasites, the muscular effect of Ro 15-5458 does not appear to be related to its schistosomicidal action. It appears that the drug requires host factors to exert its lethal effects. Conventional attempts to demonstrate bioactivation of the drug were’without effect, perhaps due to the instability of the putative active product at the experimental condition. It is also important to note that the liver homogenate system utilized in the present study may not be responsible for the generation of the active product. Also, the possible involvement of the host immunological system in parasite killing cannot be overlooked. CHAPTER IV THE ANTISCHISTOSOMAL EFFECTS OF RO 15-5458 ADMINISTRATION TO THE HOUSE HOST INTRODUCTION The in yittg studies discussed previously indicated the absence of intrinsic schistosomicidal activity of Ro 15-5458 on Sghjstosgmg mgngggi, On the other hand, the administration of a 15 mg/kg single oral dose of Ro 15-5458 to mice infected with the parasites completely clears the infection within 7 days after dosing. Thus, the drug appears to be converted to an active antischistosomal compound _i_n, v_i19_ or requires uncharacterized host factors to exert its lethal effects. Conventional attempts to detect the host-generated active product(s) or demonstrate bioactivation of the drug were not successful. Therefore, an approach was chosen in which parasites were exposed to the drug in the host before their removal for in yttrg studies. The present study utilized a form of bioassay where the survival of parasites in yitng and effects on various biochemical and physiological parameters were examined in parasites recovered from dosed mice. In most studies, drug effects were evaluated by comparison with parasites from the same group of infected mice which only received the vehicle. One problem with parasites treated in vivo is choosing when to remove them from the host to examine the effects of chemotherapy. To detect drug effects of 74 75 primary therapeutic significance, parasites have to be retrieved before the commencement of secondary morbid and host-mediated changes. For this purpose, in the _$_. [gamut-mouse model parasites are traditionally recovered prior to the hepatic shift (Jewsbury, Homewood & Gibson, 1974). In the present study, the effects of Ro 15-5458 were examined after recovery of parasites before the commencement of the hepatic shift. The specific objective was to determine the earliest detectable drug-induced damage to the parasite, with the ultimate goal of understanding the mode of antischistosomal action of Ro 15-5458. To this end, the effects of in ,ytyg treatment on vital parasite biochemical/physio]ogical processes were examined. Attempts were also made to characterize the effects of R0 15- 5458 on the biosynthesis of parasite macromolecules. MATERIALS AND METHODS A- 12m All chemicals used in this study were analytical grade and were obtained from Sigma Chemical Co. (St. Louis, MO), Mallinckrodt (St. Louis, MO) or Boehringer-Nannheim (Indianapolis, IN). Chemicals for SDS- polyacrylamide gels were from Bio-Rad Labs (Richmond, CA). Agarose (pure grade) was from Bethesda Research Laboratories (Gaithersburg, MD). [2,8- 3H]adenine (39 Ci/nlnole), [a-”P]CTP (3000 Ci/Irmole), [a-”P]dCTP (3000 Ci/nlnole), L—[4,5-’H]leucine (50 Ci/nlnole), L-[methyl-“Clmethionine (44 mCi/mole), L-["S]methionine (1088 Ci/nmole), [methyl-3Hlthymidine (74 Ci/mole) and [5,6-3H]uridine (45 Ci/mole) were from ICN Biomedicals (Irvine, CA). 76 3- mum A stock of Ro 15-5458 was prepared as an aqueous suspension in 25% glycerol - 1% cremaphor EL and a 15 mg/kg single dose was administered by gavage to infected mice 5-7 weeks postinfection. Mice infected on the same day with the same batch and number of cercariae, but which only received the vehicle, were used as the source of control parasites. C- W A 15 mg/kg single oral dose of the drug was administered 5-7 weeks postinfection to mice infected with Puerto Rican strain of 5. mgflgggi. Control and treated mice were autopsied 2 weeks after dosing and the number of parasites in the mesenteric and intrahepatic veins counted. Drug effects on hycanthone-sensitive and -resistant parasites were determined by the same protocol. Parasites selected for hycanthone resistance in the laboratory were provided by Dr. Alan Sher (National Institutes of Health, Bethesda, MD), Dr. John Bruce (University of Lowell, Lowell, MA), and Dr. Donato Cioli (Institute of Cell Biology, Rome, Italy). D. In yjtrg Qulturg and Egzgsjtg [rgnsfgr For in yttgg culture, paired adult schistosomes were isolated from mice and washed in sterile medium. Incubations were performed at 37‘C in 10 ml of RPMI-1640 medium (Gibco, Long Island, NY) buffered with 25 mM Hepes, containing 20% calf serum plus 8 ug/ml of gentamicin and 1 ug/ml amphotericin B. Triplicate dishes containing 8 paired parasites in each group were incubated up to 15 days, changing the medium 3 times per week. 77 Parasite transfer experiments were performed as described by Cioli (1976). Donor mice were treated with a 15 mg/kg single oral dose of Ro 15-5458 and parasites were isolated 1,3 and 4 days after dosing. Twenty male parasites were then transferred to uninfected hamsters (Mgsgcricetus mt). Norms were retrieved from recipient animals by perfusion 2 weeks following transfer. E. Megsgrgment of Surface Membrane Changes and Muscular Activity Parasite surfaces were scanned with a JOEL 35CF electron microscope operated at 15 kv to identify drug-induced changes in the morphology of the male parasite surface membrane. Parasites removed from the veins by careful incision and pressing were collected with a soft brush and were transferred to saline isotonic to mouse plasma with the addition of sucrose (320 mOsm). Norms were carefully washed with the same saline and were processed for microscopical examination as previously described (Neisberg gt g1., 1983). Resting surface membrane potentials were recorded from the dorsal surface of male parasites as described by Thompson gt g1. (1982). Motor activity of parasites (3 pairs/ml RPMI- 1640) was measured with a micromotility meter (Bennett & Pax, 1987). F. Mgggurgment of Parasitg th Pigment Twenty paired parasites were transferred to 250 pl of' 25 mM potassium phosphate buffer, pH 7.6. Parasites were homogenized at room temperature at 1000 RPM for 3 min in a glass homogenizer with a teflon pestle driven with a motor unit (Talboys Engineering Corp., Emerson, NJ) and 100 pl of the homogenate was mixed with 2.5 M NaOH to a final volume 78 of 2 ml. The optical density of the samples was then determined at 580 nm using a double-beam spectrophotometer (Beckman UV-5260 Palo Alto, CA). Since schistosomes contain other porphyrins that.may form ferrihemochromes that absorb at this ‘wavelength, the same number’ of parasites were incubated in RPMI-1640 medium plus 5% calf serum for 72 h at 37°C to regurgitate their gut contents (Mercer & Chappell, 1985a; Senft & Senft, 1962). These parasites were treated as described above and the resulting absorbance value was subtracted from the initial determination to account for the non-hematin absorbance. The dark pigment in the gut of schistosomes is composed mainly of hematin (Rogers, 1940). Thus, an attempt was made to compare its level in control and drug treated parasites using bovine hematin (Sigma Chemical Co.) as a standard. G. i i P t N ' P ’ nt t The weight of 30 male parasites (n=6) was determined after drying at 110°C for 15 h. Total protein was measured by the method of Albro (1975), using bovine serum albumin as standard. H. Dgtgrmingtjgn 9f Glycgggn, ATP, ngtgtg and Utilizgtion of Glugosg Glycogen was determined by comparing the concentration of glucose in neutralized aliquots of the parasite homogenate in 0.5 M KOH, as described by Tielens & Van den Bergh (1987), before and after hydrolysis with 2.2 U/ml amyloglucosidase for 30 min 'at 48°C. Glucose was measured 79 by the method of Raabo & Terkildsen (1960). To reduce glycogen degrada- tion in culture, schistosomes were transferred promptly from the veins to ice-cold medium before each experiment. For ATP measurement, 50 pairs of parasites collected in ice-cold medium were homogenized in 300 pl of equal parts of saline and 12% TCA. The homogenate was centrifuged at 10,000 g for 5 min. ATP was measured in 100 Til aliquots of the supernatant as previously described (Jaworek ' gt gl., 1974). Glucose utilization was measured as the sum of the reduction in glucose content of the medium plus the glycogen loss of the parasites during incubation. Briefly, 10 pairs of parasites were transferred simultaneously from each mouse into medium either at room temperature for in yittg culture or in an ice bath for determination of glycogen before incubation. Parasites in the ice bath were transferred to 200 pl of 0.5 M KOH for homogenization and the homogenate was stored at 49C overnight. The other group of parasites were incubated for 21 h in RPMI-1640 medium containing 20% calf serum plus antibiotic-antimycotic solution. These parasites were homogenized in the same manner as the non-incubated parasites and the level of glycogen as well as media glucose was compared to preincubation levels. Lactic acid analysis was also carried out in media aliquots after treatment with 8% perchloric acid by the method of Schon (1965). I. t f h U ake f Me lic Precursors The transtegumental uptake of metabolic precursors was determined by incubating male parasites (IO/tube) at 37”C for 5 min. Parasites were 80 incubated in Krebs-Ringers saline containing 5.5 mM of glucose buffered with 25 mM Tris-maleate to pH 7.4 (Read gt g1., 1963) for leucine and methionine or Hepes buffered RPMI-1640 medium for adenine, uridine and thymidine. After equilibration at 37°C, tritiated precursor (10 uCi/ml) diluted with cold precursor was added at the final concentration of 5X the K, (concentration of the solute yielding a half-maximum velocity of transport) value as previously determined (Isseroff gt gl., 1976, Levy & Read, 1975) to a specific activity of 33, 5.4, 44.5, 29.2 and 31.4x10’ cpm/umole of leucine, methionine,‘ adenine, uridine and thymidine, respectively. After incubation, parasites were washed 3 times with cold medium and transferred to a glass vial containing 2.0 ml of 70% ethanol and extracted for 24 h. The amount of precursor transported was calculated from the specific activity at the start of each incubation. J. .-. r-i- vf t - 1,0 .. .t'u of -- i ‘ in . Pa asi - P otein To determine the incorporation of [’H]leucine into parasite proteins, 60 male parasites were incubated in minimum essential medium without leucine, buffered (pH 7.4) with 25 mM Hepes containing 20% calf serum and 400 “Ci of [’H]leucine (50 Ci/nmole). Following 24 h incubation at 37°C, parasites were washed and then frozén and thawed to remove the surface membrane. Detegumented worms were homogenized in 1.0 ml of 0.05 M Tris buffer (pH 7.5) and centrifuged at 100,000 g for 45 min. The resulting pellet (100k pellet) was solubilized for 10 min (3% SDS in Tris buffer at 100°C) and proteins (60 ug/well) were separated using 7.5% SOS-polyacryl- amide gel electrophoresis (Laemmli, 1970). Five mm fractions of the gel were cut, dried overnight at room temperature and solubilized in 30% 81 H,O,:NH,OH (99:1) at 60°C for 5 h and counted with a Beckman LS7600 liquid scintillation spectrometer after addition of 100 pl glacial acetic acid and 10 ml of ACS aqueous scintillant (Amersham). K. W Isolation of nucleic acids was performed essentially as described by Chirgwin gt g1. (1979). Parasites were ground into a paste in liquid nitrogen and the paste was transferred to 4.4 M guanidine thiocyanate containing 0.9% fi-mercaptoethanol. After I h of denaturation, the homogenate was layered on a 5.7 M and 3 M preformed CsCl step-gradient and centrifuged at 29000 rpm for 22.h in a Beckman SH41 rotor at 19%:. Livers of uninfected mice were extracted simultaneously for comparison in the same manner. The DNA at the CsCl interface was collected with wide- bore pasteur pipets and dialyzed against 10 mM Tris HCl, pH 7.5, 1 mM EDTA and 0.1% SDS for l h. The DNA solution was made 50 pg/ml in DNase-free RNase A (Sigma Chemical Co., St. Louis, MO) and dialyzed for another hour against 10 M Tris HCl, pH 7.5. Proteinase K (Boehringer-Mannheim, Indianapolis, IN) was added to 50 pg/ml and the dialysis continued overnight with several changes of the dialysis buffer. The DNA solution was then extracted 3X in equal volumes of Tris-saturated phenol, 2X in chloroform and 2X in ether. At this point, the solution was transferred to acid-washed Corex tubes and precipitated with 2.5 volumes of ethanol and 0.5 M ammonium acetate, pH 5.5, and stored at -20°C. The RNA at the bottom of the tubes was suspended in diethyl pyrocarbonate-treated water and extracted 3X with butanol/chloroform (1:4) before storage. Poly(Af) 82 RNA was isolated using Oligo(dT)cellulose (BRL, Gaithersburg, MD) as described by Jacobson (1982). L. W Total RNA (10 pg), after checking for integrity by electrophoresis on a 1.2% denaturing agarose gels, or 1 pg poly(A’) RNA was translated in a rabbit reticulocyte system according to the supplier’s protocol (Promega, Madison, NI). Translation was measured as the incorporation of E”S]methionine (1088 Ci/mmole) in a 2 pl aliquot of the 25 pl translation mixture as alkali-resistant, TCA-precipitable counts. Samples that incorporated label 5-fold or more over zero-message controls were loaded on 10% SOS-polyacrylamide gels (Laemmli, 1970) after 5—fold dilution in sample loading buffer containing 2% SDS and heating at 95%: for 2 min. After electrophoresis, gels were fixed and stained with 0.02% Coomassie brilliant blue R in 50%.methanol, 7%.acetic acid overnight. The gels were dried, prepared for fluorography with EN 3HANCE (New England Nuclear) and exposed at room temperature to Kodak X-OMAT x-ray film for 3-5 days. The resulting fluorograms were quantitated with an LKB UltraScan XL Laser densitometer (Pharmacia, Piscataway, NJ). M. R in l smid n obe S nt e i Bacteria harboring plasmids with schistosome ribosomal RNA genes (rDNA), pSM r3.0 and pSM r4.7 (Van Keulen gt gl., 1985), and superoxide dismutase (SOD), pSM 12.38 (Simurda gt g1., 1988), were obtained from Dr. Philip LoVerde (SUNY-Buffalo). The insert of pSM r3.0 is 3.0 kb and contains the coding region of the 285 rRNA gene. The insert of pSM r4.7 83 ' is 4.7 kb and contains a sequence that codes for the 185 rRNA. pSM 12.38 contains a 320 bp insert that encodes the carboxy terminal of schistosome SOD. The recombinant plasmid containing sequences for the fi-actin gene was provided by Dr. Harold Neintraub (The Fred Hutchison Cancer Research Center, Seattle, HA). The insert (ca.l kb) is from chicken brain (Cleveland gt 31., 1980) and is subcloned into the Sma I site of plasmid pSP64 in the antisense orientation. Bacterial culture and plasmid preparations were performed using standard methods (Davis gt gl., 1986). B-actin riboprobes (cal 10' dpm/pg RNA) were synthesized after linearization of the clone with Pvu II in a transcription system contain- ing 50 pCi of [a-“PJCTP (3000 Ci/mmole) according to the manufacturer’s protocol (Promega, Madison, WI). Random primed synthesis of the ribosomal DNA and SOD probes was performed as described by Feinberg & Vogelstein (1983) after electroelution of the bands of interest from agarose gels (Figure 14). Probe synthesis was performed using a kit obtained from Boehringer-Mannheim (Indianapolis, IN) with 50 pCi of [a-3’P]dCTP (3000 Ci/mmole) as a labeling nucleotide to a specific activity greater than 10‘ dpm/pg DNA. N. Qgt-Blgt Analysis Total RNA (5 or 10 pg) was denatured with 1.2 M glyoxal in 10 mM phosphate buffer, pH 6.8, by incubating for 1 h at 50°C. Suitable dilutions of the samples in 0.1% SDS were applied to Zeta probe nylon membranes (BioRad Labs, Richmond, CA) using a manifold (BRL, Gaithersburg, 84 u u use .o.me 2mg u m .~.¢c 2mg n < 8w.o :o cmscomema ma: comumeaamm .mm.- 2mg .vmms ma: _wm ae_ a mean: mm.- zmg com unwuxo pom amocmmm .mwmweozaosuuw—m mgommn Amm.~H Zmav ~ hm; vcw 84”L 2mg ucm ~.¢L :mav “zoom gum: umpmmmwv mm: comuuacuxm Eugen cm>_m mmaos a seem emcm>oumc mew: www.macmm mcpmou gmuma Hmmwmma .w m—me mo muamezm pameou use we mgaaemocups cceuuwpm mcwccmum .emcz age .mg ae=m_e EA ”rag/‘54 5‘ (...; .. A «a» ,a'flo 0‘73 3 15Ku-xs4a .5' 1 rs. ., i.m _ é . ~*? 33 ‘£:“. 35 ’ ‘5’ 29 ii"; . ‘ . . . .J .7” 2,4 . «1 f2}; ' 13? ~43“. . 2'73. 21.}: ”J ‘ _" '9319 10 an ceosri Figure 15 94 .2525 8.3. 5828 so: 283:; 328:??? 528.2338 “53:26 e ea 3. .+. :3... 2: 9; 333m 63339:. 2.5 Lou Sigma 9; 53232.33 mic cote 258:3... 3...: 9:. as 3:; so: 33.5.. 98: 323.2:— cm..:& 2.133 .w .3 532693 mam out» 5. 2: .8 3.3.2 3. 35325.5; a»? 3. me .883 .2 953... ... Nb 3 *N z N. s m .300 ._ . .8. -omp .. DON - 0mm sKop g/agowaysfifig TABLE 13 Effects of R0 15- 5458 on Gut Pigment, Glycogen and ATP Content of S. mgnggni‘ Day of Parasite Gut Pigment Glycogen ATP Recovery (pM) (pg/male) (nmoles/pair) Control 1.7 10.20 12.710.26 1.210.2 (0.4910.06) (420110) (39.016.1) l 2.4 10.11° 12.310.23 N.D.° (0.7310.01) (4301 8) 2 2.0 10.20 13.110.90 N.D. (0.6310.06) (480123) 3 2.010.01 12.410.25 1.110.4 (0.65) (450110) (36110.0) 4 1.8 10.16 11.410.50 N.D. (0.6210.05) (480128) ‘Results are expressed as the mean of 2 experiments 1 SD (n=20). ANOVA was performed to determine the significance of differences between the means. I’Significantly different from control group, ANOVA (Newman- -Keul’s), p<0. 05. °Not determined. NOTE: Results shown in parentheses are gut pigment (pmoles/mg protein), glycogen (pg/mg protein) and ATP (nmoles/mg protein). 96 TABLE 14 Effect of Ro 15-5458 on Carbohydrate Utilization and Lactic Acid Production of S. mgnggni 3 Days After Dosing Carbohydrate Utilized Lactate (nmoles of glucose/pair/h) (nmoles/pair/h) Medium Glycogen Total Control 681 3 1814 86.714 8710.1 (1.810.1) (0.4610.01) (2.210.1) (2.2310.01) Ro 15-5458 70112 1114‘ 8018' 7310.1“ (2.110.2) (0.3 10.01) (2.410.3) (2.1510.01) Values are the mean 1 SD of 2 experiments (n=14). ‘Significantly different from controls (ANOVA, 0.1 g p g 0.05). NOTE: Values shown in parentheses are nmoles/mg protein/h (Mean 1 SD). 97 Hematin forms a ferrihemochrome, characteristic of porphyrins with either -OH or'FhO on coordination position 5 and 6 in alkaline solution. A linear relationship (r-0.99) was observed between the optical density at 580 nm and the concentration of bovine hematin in the range of 1 to 100 pH. The result of gut pigment level comparison experiments showed no reduction in the gut pigment content of treated schistosomes. He actually observed significantly'elevated pigment levels for*parasites recovered one day after treatment (Table 13). A drug-induced loss in total protein content and biomass of parasites was observed 3 days after dosing (Table 15). Male parasites showed change before females which was significantly lower for both sexes at later times of parasite recovery. Because parasites lost protein at the time when no effect on glycogen content, ATP and gut pigment was apparent, the uptake of some important metabolic precursors into parasites retrieved 3 days after dosing was evaluated. The rate of uptake of leucine, methionine, adenine, uridine and thymidine was significantly reduced in drug exposed parasites (Table 16). When the incorporation of'[°H]leucine, [3H1thymidine or [’H]uridine into TCA insoluble fractions of the parasites was determined as previously described (Chapter 111) using parasites recovered 1, 2 or 3 days after dosing, it was found that the incorporation of leucine and thymidine, but not uridine, was significantly reduced (p<0.05) for parasites recovered 3 days after dosing compared to untreated parasites (Figure 17, panel A). 98 TABLE 15 Effect of Ro 15-5458 Treatment on the Protein Content and Height of 5. 1112115291 Days After Protein/worm‘ ' Neight° Dosing Male Female Control Ro 15-5458 1 9612 (3) 10013 (3) 0.14310.02 0.13710.04 ‘ (n=6) 2 9215 (3) 9814 (3) N.D.‘ N.D. 3 7418b (5) 8312b (2) 0.12410.03 0.10310.01° (n=6) 4 5819b (3) 6517b (3) N.D. N.D. ‘In each experiment, control and treated parasites from the same group were used in parallel for protein measurement. The protein contents of control groups taken as 100%, were used for comparison. Results (percentage of untreated control) are mean 1 SD of the number of experi- ments shown in parentheses. bSignificantly different from controls, p50.05 (ANOVA). °Values are dry weight, mean 1 SD, in mg/male. °Significantly different from control group (Student’s t-test, ng.05). ‘Not determined. 99 TABLE 16 Effect of Ro 15-5458 on the Uptake of Nucleic Acid and Protein Precursors of S. mansgni Recovered 3 Days After Dosing Precursor (mM) Control Ro 15-5458 Adenine (0.25) 0.0261.003 0.02010.001‘ Thymidine (1.0) 176123 124129‘ Uridine (1.0) 132142 89125‘ Leucine (0.0015) 1815.0 1514.2‘ Methionine (0.0015) 8.112.0 6.911.2‘ ‘Significantly different from control group, 0.1 < p < 0.05 (ANOVA). Values are uptake rates, mean 1 SD, in nmoles/male/hr, for 2 experiments (n=20). Uptake values are not corrected for diffusion. 100 .87... name 3:. .o co.ume.o.mca.a .oo. m.m=cm .m..~ ems.c..ma mm: «mm.-. m.ucmu=.m .mo.owa .mpo..=ou 8.. 8.3.3.9.... 328...??? 33.3.2. 3.3... ... 3.3.88.6... m... 3.328 8 33 m3: .xoo. mm :mxau .mpo..=oo mama. so.. .:.m.o.a ms\sau. mmzpa> mmm.m>< .ucmsm.=m~ms xu.>..u~o.u~. .... 33 3.33 .38 35...... 3.5m m... .... 8.8: 3.3... 33.3.. m... 3383. 8...... 8.... .8... 3.963. 383.8 83......33 83 ... .323: ... 8383.. 3 2.882.... 5.: 8:38. .8: mm..ma.~a .o a:c.m mama as. :. um.=m~me we: a..>.uumo.um. mpaspom=.- can mpnapom-u.ua .m .mcma c. .2 3.5.: 3.3883... 338.33.933 3 8.83... 33 8.3.8.8... ....a 8.3... ...... 8.3.52. .888. ..8...fi.u .... ....)u: o. 8.53:8 ......3... 325%... ... .. . .... 33...: m3... 8.3.. .8: 3.82.... 3....» m... an 8.... :5... .8383. 38.3.3 43%.... .w .... 33.8... 33385.32. .33 m... 8... 38.23... 3.3.... .3 8.3.8.8... m... ..o 33.2 3. u... 83.: .2 3.8.... 101 s. m.=a—. coon 0.9.? o .050 >338. 3.3.2. .0 «.80 w m .. w ... 2.39.4 . .4 26.5....- II 2.25 0......- 4 .4. O N .- é é é (gonna: p ”amen Bw/Ludp I ' 102 The specificity of drug action on the incorporation of leucine was tested by comparing the radiolabeled proteins separated by electrophore- sis. Ro 15-5458 treatment resulted in reduced incorporation of [fli]leucine into the 100k pellet proteins of male 5. mansgni retrieved from mice 3 days after dosing. A 45-50%lreduction in the incorporation of leucine was observed for proteins ranging in apparent molecular mass from 36 to 96 kD (Figure 18). Drug effect on the incorporation of leucine preceded the drug-induced reduction in the internal pool of the amino acid (Figure 17, panel B) suggesting the possibility of drug affecting the synthesis of parasite proteins. Prompted by this result, RNA.extracted from parasites retrieved from mice 20 h after dosing with Ro 15-5458 or vehicle was translated in a rabbit reticulocyte in litrg translation system. When 10 pg total RNA from each group was translated, a significant reduction (p<0.05) in the incorporation of (”Slmethionine was observed for RNA from treated parasites (Figure 19, panel A). However, RNA extracted from uninfected yet treated host liver supported translation to the same degree as RNA from non-treated livers (Figure 19, panel B). In addition, a 75 mg/kg single dose of the inactive structural analog, Ro 21-6787 was admini- stered, to infected mice and RNA extracted from parasites recovered 48 h after dosing. RNA extracted from these parasites supported translation as efficiently as control parasites, while RNA from parasites treated with 15 mg/kg Ro 15-5458 exhibited a time-dependent reduction in the incorpora- tion of [”S]methionine (Table 17). On the other hand, when the same amount (1 pg) of'polyKAV) RNA from control parasites and parasites 103 0—0 Control . I l l l l l I I I ' j o 2 4 6 a 10 12 14 Fraction Number Figure 18. Effect of Ro 15-5458 on the incorporation of [’Hlleucine into the 100K pellet proteins of 5. m1. Male parasites were incubated in m in leucine-free medium containing 20 pCi/ml [°H]leucine. Incorpora- tion was measured in gel fractions after solubilization as described in the Methods section. 104 .m.m»—~:~ =c_mmm.mo. .~m=.—-:o= an ace... «.mz mm>.=u ..nucv :o..uu.=. .o mmsoaan a:m.m...u so.. <2: m=.m= cma.c..aa «.mz mu=m5_.maxm .m_o..=ou umammma-o.m~ .¢>c «_mmgaczm .o =o..a—:s..m m.~ .am H gums. ascgm ma—zmmz .mmmmgacm.~a :. cm.ao.u=. awe.u as awe; as. so.. cm>osm. mm..ma.aa vauau.a-m:.u .o 2.28 ...... 2:28. 3: <2: .m2=8 3.33.5-5» .2353.-:3:a ... 22.6.38... .8 825...: 35a 2 8.6.5. n.2,. 32.5... t. u ....m .32.»... 3.3583... 5.3... p: 3 a 5 823...... mm: <2: page. we a: cup .__ mazes .o .< _ocaa. mscmoam.sum .c ca...eua as. .aa.a 33...: 252—2.... 5 232... ..2. «5.5.3333. 2 8.2.8.85 m5 .2 3.38 as: ...: 8:5“. A 32...: v ma: A .8353 v u}... 8 8 3 on o 3 8 3 as o 1 I _- F .. .n .. . a ... a: .... a... .. 2 as a. uni . .8380 .2280 m < . n momma: Mani-a 105 TABLE 17 in Vitro Translation of RNA Isolated from §. mansoni “S-Methionine Incorporated Stimulation Rela- Source Of RNA. (cpm x 10'")b tive to zero Message Control 15.2 5.6 Ro 15-5458 (6 h) 16.5 5.1 Ro 15-5458 (12 h) 8.7 3.2 Ro 15-5458 (48 h) 7.3 2.7 Ro 21-6787 (48 h) 15.0 5.5 No RNA 2.7 1.0 ‘RNA was extracted from control or drug-treated parasites and translated in a rabbit reticulocyte system. Parasites from treated hosts were retrieved after the time shown in parentheses. bResults are alkali-resistant TCA-insoluble counts in a l-ul aliquot of the reaction mixture. 10's recovered 3 days after dosing was translated, the same efficiency of translation was observed (Figure 20). Electrophoresis and subsequent fluorographic analysis of the translation products showed a similar pattern for both treated and control parasite mRNA (Figure 21), with subtle quantitative and qualitative differences (Figure 22) as determined by densitometric measurement of the resulting fluorograms. The level of some low molecular weight (Mr) proteins was reduced in products directed by mRNA from treated parasites (stars in Figure 22). One striking observation in these results is that higher Mr peptides were synthesized in more abundance from treated parasite mRNA than from control mRNA; Since the.same amount of total RNA from Ro 15-5458-treated parasites was less efficient than control RNA in directing peptide synthesis, the possibility that the drug reduced the relative amount of mRNA was tested. ' Thus, first the effect of treatment on the total RNA content of adult schistosomes was examined. The same number of control and treated parasites (500-1000), were used for extraction and the resulting RNA compared by measuring the optical density of aliquots of the samples using a.double-beam spectrophotometer (Beckman UV-5260, Palo Alto, CA). Admini- stration of a 15 mg/kg single oral dose of Ro 15-5458 to the host resulted in a time-dependent reduction in the RNA content of parasites (Table 18). A 14%, 30% and 41% reduction was observed for parasites recovered from mice 12, 72 and 96 h after dosing, respectively. 107 1 2 3 4 RD zoo— "' 116— 97— . ‘ 66— 42— “7" .. ;;;; 21-‘"‘_ Figure 20. Fluorogram from the j_n_ yitrg translation of poly(A’) RNA. Translation products of 1 pg poly(A’) RNA from control schistosomes (lane 1) and schistosomes recovered 3 days after dosing (lane 2). Lane 3 is from 10 pg total RNA of control paraSites and lane 4 is without any added RNA. 108 Figure 21. Fluorogram showing the polypeptide pattern of the _m vjtzg 'translation products of schistosome and liver RNA before and after treatment with Ro 15-5458. RNA from control schistosomes (lane 1) and schistosomes recovered from the host 12 h (lane 2) and 72 h (lane 3) after dosing as well as control mouse liver (lane 4) and liver from mice sacrificed 8 days after administration of a single 15 mg/kg dose of R0 15- 5458 (lanes 5 and 6) were translated in a rabbit reticulocyte system. The products were separated on a 10% SDS-polyacryl amide gel for fl uorography. 109 .—c..=ou :— =oom so: was: ~.uxa u :o..< .—o..:ou o» cu.aqzou a:o.m gag. c. .mp—asu a. ecaa u .aam .amem-m_ a: a..3 a=.mou .u..a aunt n n u uza .; N. vu>¢...m. m¢u.ma.aa n a .a. <2: game m.~ ma=.a> .co..-.u..a.g .c no...m=o.=. m>.ua—m. .ou vo.~aeou «.mz «mac—m age can m—asam sumo .a. was.cm.m: was <2: .o acacia .m> ago as. .o =a_mmo.mm. .ao=.. .<2: :.au~ o. 2.aa=msmpqscu one.:oa.. uc—mna—-:= a 2a.: co~.v..222 tea moca.naoa cap»: a. no.paaa a.u3 :38.: 28am so.» Am: m.m.o can .m~o.o .m~._ .m.~. <2: .8 macacsa .ga.a...= .qaqwama .w .o .ga.=ou <2:: =..ua «2. ca .cma.am.. «...-.. o: .c .ua... .m~ o.=a.. l v .IIJuAU 0:9:0“ 3:93:91 VIII/[IA v :0 I I CD 0 P in :5. cu c. 8 anemia. 0: .3... .N. .228 .2: i .. a. 83.: 0: ...om .H. .. mm 83...... 0: ...om .H. .. .. a. Santa. 0: ...om an. . .. ..u anemia. 0: .5... a ..u. 83...... 0: ...om I 2 .828 ...8 .H. \Y\\\\\ \X\4 l_ I (sigun 7001;!qu ) (anal VNEW-I ”113V 0. n 113 .mo.cv. ..<>oz<. m—o.u:cu so.. u:m.m...u 2ppmu.um.u~.m« .Amucv cm H mpm>mp <2: cams m.~ mm=p~> .cc.am~.u..nxg mo 83.2.35 2.33... m... .8 8.2.28 m8... 887 ...... was... 88 .8 82.8.2. m2. <2: we asses. .m> .o.o .o co.mmm.mm. .~m=.. .o.m. 2m: .o :.e. 2m: .mgu.m 2a.: umno.a can mm=~.nsms :o—2: :o uw~._.ooes. new Am: m~m.o .m~.. .m.~ .m. amen—.2 appm..mm m.wz mmpasam <2: vm.=a~=mo .5898 .w .... 226— <2:. mmN ...... mm. 2: .... “8.58... 38-..: o: .8 88.5 ...N 9.35... 114 cu a.=m.: OH... 2m... 5.... Ema. 0.— ON 0.0 o... 06 ... at n. a: .3)... .N. o o .0538 ...o>... .MH. ... n n. o: .8 a ... u. n. a: .5... ' .o....oo ...om .HU (suun Momma) SIS/\al (my 115 l 2 3 4 5 6 Figure 25. Ethidium bromide-stained RNA gel. 1.2% agarose gel containing 0.7 ug/ml ethidium bromide was used for RNA separation. RNA is from control schistosomes (lane 1), or schistosomes recovered 12 h after dosing with Ro 15—5458 (lane 2), 3 days after dosing with Ro 15-5458 (lane 3). RNA on lane 4 is from control mouse liver, while lanes 5 and 6 are from Ro 15-5458-treated mice sacrificed 8 days after dosing. 116 both messages, it appeared that the 1.9 kb species was affected to a greater extent (Figure 26). This was confirmed by electrophoresis and northern analysis of 1 pg poly(AF) RNA from control and treated parasites retrieved from the host 12 and 72 h after dosing. The level of the 1.4 kb actin mRNA was dramatically reduced in parasites recovered 3 days post- dose (Figure 27). A mRNA (ca. 0.78 kb) that hybridized with the SOD probe from schistosomes was also reduced after treatment with Ro 15-5458; a 12 and 44% reduction in the content of SOD mRNA was observed for parasites recovered 12 and 72 h after dosing, respectively (Figure 28). Figure 29 depicts the autoradiogram and ethidium bromide stained. gel picture from the same experiment. The RNA was transferred and simultaneously hybridized to both the p-actin and 18S rRNA probes. Result from this experiment indicated that treatment lowered the level of the rRNA’s in light of the amount of RNA applied to the gel as can be evidenced from the ethidium bromide-stained pictures. To check the possibility that Ro 15-5458 treatment altered the restriction fragment pattern of parasite DNA, a comparison was made between DNA extracted from control and treated parasites using ”P-labeled probes that hybridize to either of the DNAs that code for the mRNAs described above. Four to eight pg of genomic DNA was digested to completion with either EcoRI or Hind III restriction enzymes. The resulting fragments were electrophoresed and transferred and each filter was sequentially hybridized with probes for SOD, actin and rRNA by stripping the filter in water containing 1% glycerol at 85°C and rehybridization. Hybridization 117 .maopg .mn ma czosm m.m s~.mo.um.ou:~ as. :o mam. gumm so.. <2:s .o mm~.m gees mo co..-.n..n22 mo mwpa.mcmac. m>.ua.m. mgh .m.mx.as m~.m ma awn: m.m3 .m.m=~.u m.o.mn um:.~um magma <2: .msomon.. muse: ..nx. mm~.m o» .mum. m.maE:2 ..m mcmp. ; m. we. .8 mg... g N. ..m as... g .N .8... 88.2 um.....-mmem-m. 6. 5a.. um.m>oum. “8.....aa 8:. .N wasp. mops umaam.a-:m:m-.~ o: so.m 2 N: um.m>oum. mmu.m~.aa ... camp. mm._m~.~a po.ucou so.. cmuapom. no: <2: .m_gumg mmu_m~c~a mum u can < mpmcma .o mead .mmcm-m_ om gap: mzpmou gmuma Am pmcma .e wcap mu new < mpmcmn .m ocapv : om new .Am pmcma .m mcmp no new < mpmcma .v mcapv ; - ..u new < «Focaq .m mcmpv ; vN .Au new a .< mpwcaq .~ mcapv ; N“ um>m_gamg mma_m~saa umuamsa .Au van m .< «pagan .~ mcapv mmEOmcum_sum pccaccu.sogm umaapom, mm3._u=_ ca go mama—a use =. =o_uagu:ou=ou as“ mucommgaog —ona»m :uau 3.5: ... ..n ..8 u...o.......m.~.m..... . . m « .... 4 D m 4 a .93 m Q. mw .oonmum .I NWm C .8. ...." d 4 a :3: m «mo: _ago c_m:.m ax\ms cm a we =o_uagun.=¢ac<.m=u _ m:_3o——om mu_aa~¢ ea asmapa as» c. omem-m_ ex ea =o.aasu:ou=ou _ x_azmmm< APPENDIX II Pharamcokinetic Parameters Obtained After Fitting the Plasma Level-Time Data to a One-Compartment Model with Either First-Order Absorption or Zero-Order Absorption Using the PCNONLIN Program Parameter Volume of distribution Apparent absorption rate constant Apparent elimination rate constant AUC C... Correlation (r) First-Order Zero-Order Model Model 251473‘ 47.715.2 (-175-I75') (35.6-60) 0.461861 --- (-320-320) 0.461862 0.2110.06 (-200-200) (0.09-0.34) 4.411.3 4.910.93 0.90 O.7410.O9 0.92 0.96 'Val ue 3 SE I'95% confidence limits 160 APPENDIX III EcoRI (A) and Hind III (8) Restriction Pattern of DNA Isolated from R0 15- 5458- treated S. mgngoni 91o 11 12 <-5-5 +3 .0 DNAs were isolated from control parasites (lanes 1 and 7), parasites recovered from Ro 15-5458-treated mice after 12 h (lanes 2 and 8), 24 h (lanes 3 and 9), 72 h (lanes 4 and 10) and 96 h (lanes 5 and 11). Lanes 6 and 12 are DNA from parasites exposed to Ro 21-6787 (15 mg/kg) in the mouse and retrieved 48 h after dosing. Numbers at the arrow refer to sizes (kb). 161 APPENDIX IV Restriction Map of rRNA Gene from S. manggnj ikbp 185 285d 28 so — _— H P 'H'E HB 85 HEHSol SLOIEIIL! 13?? EH [14 11 I 1911 1111 Hmdm '5 30 ‘5 zs 4'? 1.9 “1141’ 15 Born HI . 1.1 1,1 : r 25 1.2 23 F ‘ 1: 11- 1 Eco R1 47 1.6 3.0 Reproduced from Van Keulen gt al., 1985 (Mol. Biochem. Parasitol. 15,: 215-210). 162~