Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. · . STUDIES OF THE PHARMACODYNAMICS AND MODES OF ACTION OF ANTHELMINTIC DRUGS A THESIS PRESENTED IN PARTIAL FULFILMENT OF THE REQU IREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT MASSEY UNIVERS ITY U MIN SOE 1977 ii Abstract' of a thesis presented in partial fulfilment of the requirements for the Degree of Doctor of Ph ilosophy STUDIES OF THE PHARMACODYNAMICS AND MODES OF ACTION OF ANTHELMINTIC DRUGS by U MIN SOE 'ltl,e aim of this work is to extend existing knowledge both with respect to the mode of action of anthelmintics and the biochemical and physiological mechanisms which may be disrupted by drug action . The helminth species examined include nematodes , Ascari s suum, Ascaridia ga lli and Tr ichuris ovi s and cestodes , Moniezio" T. hyda tigena , T. taeni reformis and Echinoccocus granulosus; the anthelmintics studied were methyridine , diethylcarbamazine , pyrantel , morantel, tetramisole , levamisole , dichlorvos, vincofos , cambendazole and mebendazole . The helminth characteristics selected for most intensive study are (a) the occurrence and properties of helminth cholinesterase and (b) the uptake of glucose. The breadth of the study was limited by the availability of fresh material and not all combinations of helminth and drug were investigated. The histochemical localisation of cholinesterase activity in whole mounts and sections of tapeworms using thiocholine esters revealed a complex network of tegumental receptors feeding a nervous system with efferents to suckers , rostellum and hook mu scles. It is suggested tha t tapeworms have re flex arcs involving these struc tures allowing them to maintain their position in the host intestine in spite of peris taltic action . Th ese arcs are susceptible to anticholinesterase anthelmintics . Other cholinesterase activi ty is associated with the scolex , cirrus , genital pore and sometimes the tegument. High cholinesterase specific activities against acetylthiocholine were measured in Echinococcus scoleces and tapeworms , but lower leve ls in nematodes. Differential centrifugation of homogenates was used to iii study their occurrence in the tis sue and facilitate further characterisation . However, the enzyme was widely distributed in these species although somewhat higher in the particulate fractions. Ac tivity was increased little , if any, by attempts to solubilise it with the detergent, Triton X-IOO. Cholinesterase in some fractions particularly from T. ovis , had a high tempe rature optimum around 600C, but never showed the phenomenon of autoinhibition by substrate at concentrations up to IO-2M. Cholinesterase in species of worm with high levels of en zyme was more sensitive to eserine inhibition than those with lower levels. In studies of glucose uptake from the medium by As caris and two tapeworms , it was confirmed that transport into Ascaris was strongly inhibited by certain benzimidazole anthelmintics. Transport into Ascaris , bu t not the cestodes, was also discovered to be sensitive to local anaesthetics such as procaine or lignocaine . Uptake into tapeworms was inhibited by the absence of sodium ions , phlorizin, iodoacetate and dinitrophenol. It was less inhibited by benzimidazoles and not at all by organophosphate anthelmintics , but was sensitive to phenolic drugs such as hexachlorophene and nitroxynil . In the dog and sheep , a number of anthelmintic drugs administered intravenously showed predominantly nicotinic effects on blood pressure and respiration supporting the cholinergic action of these drugs . Although sheep red-cell cholinesterase is more sensitive to inhibition than that of all helminths tested , the oral route of administration of anthelmintics remains safe for the host and effective against intestinal parasitic worms . iv ACKNOWLEDGMENT I wish to express my gratitude to my chief supervisor D1·L. S. Forbes, who proposed the area of research covered in this thesis and for his suggestions and criticism and for the arrangements which were made for bringing this thesis to completion . I am greatly indebted to my other supervisor , Dr.R.M. Greenway for hi s role in supervision , continued interest, and never-failing support and encouragement. I have much pleasure in expressing my grateful thanks to Professor R.E. Munford , Head of the Department of Physiology and Anatomy , Massey University, for his computing and statistical analysis and the provision of laboratory facilities. I wish to express my gratitude to Dr. D.O. Heath and staff at Wallaceville Animal Re search Centre , We llington , for supplying T. ovis cysts and for valuable suggestions. I am indebted to Mrs Sharon Pickett for her technical assistance and typing of my manuscript and Mrs Fay Wicherts for her careful final typing . The enthusiastic technical assistance of Messrs R.N. Ward , B. O'Sullivan , and R. Te lfer and our photographer , Mr T. Law are warmly appreciated . Acknowledgements are due to the staff of the library , especially Miss E.M. Green, from the interloan section of the library at Massey University , for facilities in verifying the references from different libraries throughout New Zealand and overseas . This investigation ha s been financially supported by the Burmese Government and the New zealand Government under bilateral aid arrangement of Colombo Plan Scholarship and I am much indebted to both Governments . Finally , I thank my mother Daw Kyaw Shin , who was deceased during the course of my studies , in Burma , for her upbringing and persistant encouragement and then my wife Kyi Kyi Nyunt and our son , Soe Wunna for their encouragement during our separation for the five years while I was engaged in this research in New zealand . v CONTENTS Page No CHAPTER 1 The significance of helminth infections and their control 1 CHAPTER 2 M9de s of action of anthelmintics 6 CHAPTER 3 Pharmacodynamic studies related to anthelmintic action and their effects on host cholinesterase 37 CHAPTER 4 Helminth cholines terase and the influence of inhibitors and anthelmintics 77 CHAPTER 5 Helminth glucose uptake and the influence of inhibitors and anthelmintics 180 CHAPTER 6 Helminth cholinesterase: Histochemical studies and the influence of inhibitors 241 GENE RAL DISCUSSION 285 REFERENCES AND ADDENDUM 287 1 1.0 CHAPTER 1 THE SIGNIFICANCE OF HELMINTH INFECTIONS AND THEIR CONTROL 1.1 THE GLOBAL SIG NIFICANCE OF HELMINTH DISEASE 1.2 CHEMOTHERAPY AND ANTHELMINTICS 1.3 HELMINTH CONTROL BY DRUGS 1.4 DEVELOPMENT OF NEW ANTHELMINTICS 1.5 SIGNIFICANCE OF THE PRESENT RESEARCH / 2. 1.1 THE SIGNIFICANCE OF HELMINTH INFECTIONS AND 1. THE IR CONTROL 1.1 THE GLOBAL SIGNIFICANCE OF HELMINTH DISEASE Because of the need for brevity only a few generalisations can be made concerning the overall relation of helminth disease to world health and live stock production. In man , the major problems are undoubtedly located in tropical and sub-tropical areas where climatic , ecological, sociological and other factors combine to favour persistance and spread of parasite helminths , and in large areas of the world almost all the indigenous populations may be infected with such serious and debilitating conditions as bilharziasis (schistosomiasis) and hookworm disease (Wilson and Schild , 1959) . The topic has been dealt with dramatic­ ally in the classic treatise 'This Wormy World ' (Stoll , 1947) . Some relevant data are given in Table 1.1 TABLE 1.1. SHOWING THE EXTENT OF SOME HELMINTH INFECTION IN MAN * Infections Number of infections References (x 106 ) Total number of infections 2250 Stoll (1947) Number of people infected 800 Ditto Ascaris lumbri coides 644 Ditto Hookworm 456 .8 Ditto Tr ichuris trichiura 355 Ditto Enterobius vermi cularis 208 .8 Ditto schistosoma 114 Ditto Fi lariae 304 Ditto Schistosoma ja ponicum (China ) 33 Slack & Nineham (1968) s. haema tobium and S. Mansoni 12 Ditto (Egypt) S. haema tobium (West and Central AFrica) 8.5 Ditto S. Mansoni (Brazil) 4.0 Ditto 6 * To tal world population 21§6 x 10- 3 1.3 In contrast to man, the ma in helminth problems associated with livestock production appear in temperate regions and are related particularly to deve lopme nt of highly stocked grasslands and intensive poultry and pig production units . The global economic loss arising from mortali ties, inhibi ted production and associated with development and , application of control measures, is a matter for conjecture - but must easily amount to billions of dollars annually . 1.2 CHEMOTHERAPY AND ANTHELMINTICS As in the case of infection by all small or microscopic pathogenic organisms, the main line of therapeutic defence lies in utilising the selective toxicity of suitable drugs against the pathogen. The term selective toxicity was introduced by Albert (1977) to embrace the use of chemical substances to destroy or inhibit an unwanted species (the uneconomic species) while, at the same time, causing little or no harm to a closely associated desirable species (the economic species). In medicine, the economic species may be man or some other desired animal - whereas the uneconomic species may be a virus, bacterium, fungus, protozoan, helminth parasite or neoplastic cell . In this context the use of drugs with the ob ject of causing maximal injury to the pathogen or neoplasm and minimal harm to the host is described as chemotherapy . Anthelmintics are drugs