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Factors Affecting the Anthelmintic Efficacy of Cysteine Proteinases Against GI Nematodes and Their Formulation for Use in Ruminants

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Factors affecting the anthelmintic efficacy of cysteine proteinases against GI nematodes and their formulation for use in ruminants By Wenceslaus Luoga, BSc, MSc. Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy October 2012 i ABSTRACT Gastrointestinal (GI) nematodes are important helminth pathogens responsible for severe losses to livestock industries and human health throughout the world. Control of these infections relies primarily on chemotherapy; however there is rapid development of resistance to all available classes of anthelmintic drugs, and therefore new alternative treatments are urgently required. Plant cysteine proteinases (CPs) from papaya latex, pineapple fruit and stem extracts have been demonstrated to be effective against GI nematodes of rodents, chickens, pigs and sheep. The current study extended evaluation of different plant extracts and the factors affecting the efficacy of papaya latex supernatant (PLS) as an anthelmintic against GI nematodes in a mouse model system and formulation and delivery for use in ruminants. The study started with purification and concentration of CPs in PLS using different methods to determine which of them would provide high yield of CPs. It was found that concentration by dialysis provided a high yield of active enzyme in PLS. Storage of PLS at -200C and -800C retained more active enzymes for prolonged period of time than at ambient temperature and 4oC.Motility assay conditions showed to have no influence on enzyme activity. While the in vitro experiment results showed significant detrimental effect of pineapple fruit extract, stem bromelain and little effect of kiwi fruit extract against Heligmosomoides bakeri motility. In vivo experiments showed less efficacy of these enzymes than expected when compared with PLS. i The first factor to be assessed in this study was the effect of fasting on the anthelmintic efficacy of PLS. The results showed that PLS was equally effective in reducing worm burdens whether mice were fasted before treatment or not, and by avoiding fasting the side effects of treatment were minimized. Comparison of efficacy in a range of mouse strains indicated that efficacy varied between mice of different genotype. At the dose used, the treatment was most effective in C3H mice ranging from 90.5% to 99.3% in reducing worm burdens and less effective in NIHS, CD1 and BALB/c strains (7.9%, 36.0% and 40.5% reduction respectively). However, host sex and body size were shown not to have any influence on the anthelmintic efficacy of PLS. Since CPs are particularly sensitive to pH, variation between mouse strains in gut pH was investigated but no significant differences in pH were found along the GI tract of the poor (BALB/c) and high responder mice (C3H) to PLS treatment and concurrent administration of the antacid cimetidine also did not improve efficacy. The study also explored the potential of formulation and delivery of PLS as an anthelmintic drug for ruminants. In vitro studies involving both immediate and slow release dosage formulations simulating the physiological conditions (pH, temperature and peristaltic movement) in the GI tract of the animal were conducted. In the slow release experiments, two hydrophilic matrices were tested, the xanthan gum and hydroxypropyl methylcellulose (HPMC) (both Methocel-LVCR and Methocel-CR). Methocel-CR provided better slow release results compared to the others. In the immediate release experiments 3 disintegrants (Primojel, L-HPC and Ac-Di-sol) were investigated and Ac-Di-Sol® was found to produce the faster immediate drug release rate. Preliminary in vitro studies also showed that PLS was highly effective against equine GI nematodes. ii Finally the empirical findings in this study provide useful information for improvement of formulation and delivery of these naturally occurring plant-derived enzymes for treatment of intestinal worm infections in humans and livestock, while achieving maximum efficacy and minimal side-effects. iii ACKNOWLEDGEMENT First and foremost I would like to express my sincere gratitude to my main supervisor Prof. Jerzy Behnke and my co-supervisors Drs. Martin Garnett and Colin Melia for their valuable and detailed feedback, input and ideas. I would like to thank Dr. David Buttle of University of Sheffield for advice and for answering my numerous questions on the biochemistry part of my research and to my internal assessor Dr. Ian Duce for giving useful feedback at my progression assessments. This research would not have been possible without the studentship from Mkwawa University College of Education and the University of Nottingham for accepting to host my PhD. I am greatly indebted to Ann Lowe for her friendly technical advice and long hours selflessly spent helping me in mouse dissections and worm counts. I am also thankful to Tim Smith for assisting me in the preparation and viewing of SEM specimens, and Colin Wills and Christine Grainger-Boultby for teaching me how to use the facilities in the School of Pharmacy labs. Many thanks to people of the BMSU, particular Dave who was always ready to help me with anything I wanted in the animal house. Massive thanks must go to my colleague Fadlul Mansur for his generosity and companionship throughout my study period. I would like also to thank everyone in our research group Andrew Phiri, Fawzia Shawesh, Amy Hall, Yadong Zheng and others not mentioned for their help and advice which made my life here easy. iv I owe special thanks to my wife, Irene Kasambala for her enduring love, patience and the sacrifices she made for accepting the responsibility of taking care of the family while I was away for my study. My daughters: Mercy, Lulu and Happy, sorry for missing me all this time that I was away. Finally I wish to dedicate this thesis to my parents who laid the foundation of my education using their meagre resources. v TABLE OF CONTENTS ABSTRACT………………………………………………………………………………………i ACKNOWLEDGEMENT ..................................................................iv TABLE OF CONTENTS....................................................................vi LIST OF TABLES.............................................................................xii LIST OF FIGURES ..........................................................................xiii ABBREVIATIONS............................................................................xv CHAPTER 1: INTRODUCTION....................................................... 1 1.0 Summary.......................................................................................... 1 1.1 Background...................................................................................... 2 1.2 GI Nematodes of medical importance .............................................. 2 1.3 GI nematodes of veterinary importance ........................................... 7 1.4 Control and treatment of GI nematodes........................................... 8 1.4.1 Use of Anthelmintics .......................................................................................... 8 1.4.2 Sanitation & hygiene ........................................................................................ 11 1.4.3 Rotational grazing (Livestock) ......................................................................... 12 1.5 Anthelmintic resistances of GI nematodes...................................... 13 1.6 The search for new alternative anthelmintics................................. 16 1.6.1 Animal model used in this study....................................................................... 22 1.6.2 Life cycle of model parasite – Heligmosomoides bakeri.................................. 23 1.7 Formulation and delivery of cysteine proteinases (CPs)................. 24 1.7.1 Drug formulation .............................................................................................. 25 1.7.2 Liquid-based formulations................................................................................ 26 1.7.3 Solid based formulations .................................................................................. 27 1.7.4 Tablet classification .......................................................................................... 29 vi 1.7.4.1 Immediate release tablet dosage forms ........................................................ 30 1.7.4.2 Enteric coated (delayed release) tablets ....................................................... 32 1.7.4.3 Extended release dosage forms .................................................................... 33 1.7.4.4 Technologies used for ER in livestock......................................................... 34 1.7.4.5 Factors to be considered in ER matrix formulation ..................................... 39 1.8 Hypothesis, Aims and Objectives of the study........................................... 40 CHAPTER 2: MATERIALS AND METHODS................................45 2.0 Summary........................................................................................ 45 2.1 Enzyme preparations...................................................................... 45 2.1.1 Papaya latex supernatant................................................................................... 45 2.1.2 Pineapple fruit and Kiwi fruit extracts.............................................................. 46 2.1.3 Stem bromelain................................................................................................. 47 2.1.4 Active site titration...........................................................................................
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