Jones, Amy (2011) Melarsoprol cyclodextrin inclusion complexes for the treatment for human African trypanosomiasis. PhD thesis. http://theses.gla.ac.uk/2713/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Melarsoprol Cyclodextrin Inclusion Complexes for the Treatment of Human African Trypanosomiasis Amy Jones BSc (Hons) Veterinary Science Submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy Institute of Infection, Immunity and Inflammation School of Medical, Veterinary and Life Sciences April 2011 Amy Jones© Abstract Human African trypanosomiasis (HAT) is a parasitic disease caused by the protozoan parasites T. b. rhodesiense and T. b. gambiense. The disease is currently endemic in 36 sub-Saharan countries with an estimated 60 million people at risk from the infection. The disease progresses through two stages; an early or haemolymphatic stage where the parasites are confined to the peripheral compartment and a late or encephalitic stage where the parasites penetrate the blood-brain barrier (BBB) and invade the CNS. Without treatment the disease is invariably fatal but at present chemotherapy is reliant on a small handful of drugs. Pentamidine and suramin are available for the treatment of the early stage of the disease while the CNS stage of the disease is treated with a combination of nifurtimox and eflornithine known as NECT therapy or melarsoprol. NECT therapy is only effective in the treatment of T. b. gambiense infections meaning treatment of T. b. rhodesiense infections is completely dependent on the trivalent arsenical melarsoprol. Melarsoprol is an extremely toxic compound, the administration of which is extremely painful and associated with numerous adverse reactions. The most serious of which is a post treatment reactive encephalopathy (PTRE). The PTRE occurs in up to 10% of all patients given melarsoprol. Half of all patients who develop the PTRE will die as a result of the compliciation. This gives melarsoprol chemotherapy an overall fatality rate of 5% which is unacceptably high. There is therefore an urgent need for new, safe and easily administrable trypanocides. To improve the physiochemical and pharmacokinetic properties of melarsoprol the drug was complexed with two cyclodextrin molecules, hydroxypropyl-b - cyclodextrin (HPb CD) and randomly methylated-b -cyclodextrin (RAMb CD) to produce; mel/HPb CD and mel/RAMb CD. Cyclodextrins are cyclic oligosaccharides, widely used within the pharmaceutical industry to improve the solubility and oral bioavailability of poorly soluble lipophilic drugs. In this study, the trypanocidal activity of the melarsoprol cyclodextrin complexes was investigated in-vitro and in an in-vivo CNS stage model of T. b. brucei infection. In-vitro studies showed that the trypanocidal activity of melarsoprol is retained following its complexation with HPb CD and RAMb CD. The in-vitro trypanocidal activity of the melarsoprol cyclodextrin complexes against bloodstream T. b. II brucei trypanosomes was comparable to that of contemporary melarsoprol. Furthermore, in an in-vivo murine model of CNS stage T. b. brucei the melarsoprol cyclodextrin complexes, mel/HPb CD and mel/RAMb CD produced 100% cure rates when administered orally at a dose of 0.05 mmol/kg, daily, for seven consecutive days. Contemporary melarsoprol when administered by the same route and schedule only cured 33.3% of the animals. The cyclodextrins HPb CD and RAMb CD thus increase the oral bioavailability of melarsoprol whilst retaining the compounds trypanocidal activity. An oral administrable, water soluble formulation of melarsoprol instantly eliminates the problems associated with the intravenous administration of conventional melarsoprol. Furthermore, an orally available formulation would be of great benefit in the resource poor, isolated settings in which HAT occurs as patients would not require hospitalisation during treatment thus alleviating the pressure on local hospitals. In the current investigation quantitative taqman PCR was utilised to investigate the rate of parasite clearance from the CNS during complexed melarsoprol treatment. Both mel/HPb CD and mel/RAMb CD were rapidly trypanocidal. Twenty-four hours after administration of one dose the number of trypanosomes within the brain was reduced by greater than 80% and all trypanosomes were eliminated from the brain by twenty-four hours after administration of four doses of mel/HPb CD and five doses of mel/RAMb CD. The elimination of all trypanosomes from the CNS following four doses of mel/HPb CD and five doses of mel/RAMb CD, indicates that it may be possible to reduce the dosage schedule from seven daily doses to four daily doses of mel/HPb CD and five doses of mel/RAMb CD. A short, simple, easily administrable treatment protocol is an essential requirement of any new trypanocide as if the treatment schedule is prolonged and complicated patients are unlikely to comply. CNS stage trypanosome infection is associated with a breakdown of the blood- brain barrier (BBB). Ideally following successful chemotherapy BBB function should be restored. In this investigation the effect of a curative mel/HPb CD treatment regime on the BBB was investigated in a murine model of CNS T. b. brucei infection using small bore MRI analysis. Mel/HPb CD treatment results in a rapid restoration of BBB function as by twenty-four hours after the completion of mel/HPb CD therapy the integrity of the BBB was fully restored. However, a very III mild neuroinflammatory reaction persisted in the brain for up to fifteen days after completion of chemotherapy. This suggests that the BBB damage observed in trypanosome infection may be due to either the parasites directly or their secretory products and not as a result of the ongoing neuroinflammatory reaction. Despite melarsoprol being in use for over 60 years its pharmacokinetics are poorly understood and a sensitive assay by which to quantify the concentration of arsenic reaching tissues following administration of the compound is not available. In this study a gas chromatography mass spectrometry (GC-MS) technique was developed to quantify the concentration of arsenic reaching the plasma and brain following oral and intravenous administration of the melarsoprol cyclodextrin complexes, mel/HPb CD and mel/RAMb CD. The GC-MS assay had a limit of detection of 5ng/ml and a precision (expressed as the inter- day coefficient of variation) of 13.2%. The concentration of arsenic within the brain following the oral and intravenous administration of mel/HPb CD was below the limit of quantification of the assay. The pharmacokinetics of mel/HPb CD and mel/RAMb CD could therefore not be determined in the present study. This study demonstrates that the melarsoprol cyclodextrin complexes mel/HPb CD and mel/RAMb CD are highly trypanocidal with no overt signs of toxicity and more importantly are orally available. Following the oral administration of mel/HPb CD or mel/RAMb CD the melarsoprol is slowly released over a prolonged period of time from the cyclodextrin cavity. Patients are therefore not exposed to a ‘bolus’ of the drug as is the case in the intravenous administration of contemporary melarsoprol. The slow and sustained release of melarsoprol from the cyclodextrins should result in less adverse reactions and a decreased incidence of the PTRE. Furthermore, the complexed melarsoprol treatment protocol is shorter than the currently used 10 day concise melarsoprol treatment schedule therefore the total amount of melarsoprol administered to patients will be reduced. Patients should therefore experience fewer adverse reactions. In conclusion the results from this study demonstrate that the melarsoprol cyclodextrin complexes mel/HPb CD and mel/RAMb CD are promising oral candidates for the treatment of HAT. IV “Our greatest glory is not in never falling, but in rising every time we fall” Confucius V Dedicated to mum, my shining light and inspiration x VI Acknowledgements First and foremost I would to thank my supervisors Dr Jean Rodgers and Prof. Peter Kennedy for their unwavering support and encouragement over the last three and a half years. I am eternally grateful to Jean for her constant guidance, reassurance and patience, especially over the last few months while writing up, without which I would never have reached the end. I would also like to acknowledge Barbara Bradley for all her help and support both in the lab and outside. Without her help and patience, long days in the unit would have turned into even longer nights. Thank you. A proportion of this work would not have been completed without the contributions of several collaborators. I am extremely grateful to Dr Stèphane Gibaud, of Nancy Universitè for developing the melarsoprol cyclodextrin complexes and for making me feel extremely welcome in his laboratory. At the University of Strathclyde, Department of Pharmacy I would like to thank Prof. Alex Mullen for all his helpful, enthusiastic and animated discussions and also Dr Fitsum Araya for showing me the ‘ropes’ in the laboratory. I would also like to acknowledge Prof. George Gettinby for assisting with the statistical analysis conducted in this thesis. With the help of tea and biscuits he made the world of statistics seem a less daunting place. I would also like to thank all the past and present members of the trypanosomiasis group and the department of veterinary parasitology for making the laboratory and department a pleasure to work in, and for providing an endless supply of cakes! I would like to thank all my friends and family for being a constant source of support and encouragement both through the good times and the bad. I would especially like to thank Lesley for always being no more than a phone call away. Her kind words and reassurances saw me through the low points. Finally, I would like to thank my Dad for his endless and unconditional love, support, encouragement and constant belief in me over the last few years, without which I would not be where I am today. VII Declaration I declare that this thesis and the results presented within it are entirely my own work. No part of this thesis has been previously submitted for a degree at any other institution. Amy Jones VIII Supporting oral presentations Jones, A., Rodgers, J., Bradley, B., Barrett, M. P. and Kennedy (2010). Complex melarsoprol; a possible oral therapy for the treatment of human African trypanosomiasis. Nanotryp consortium meeting. 23rd September 2010 Paris, France Jones, A., Rodgers, J., Bradley, B., Barrett, M. P. and Kennedy (2010). Complex melarsoprol; a possible oral therapy for the treatment of human African trypanosomiasis. X11th International Congress of Parasitology. 15th – 20th August 2010 Melbourne. Jones, A., Rodgers, J., Bradley, B., Barrett, M.P. and Kennedy, P.G.E (2010) Complex melarsoprol: a promising candidate in the treatment of human African trypanosomiasis. Faculty of Biomedical and Life Sciences Seminar Series. Jones, A., Rodgers, J., Bradley, B., Kennedy, P.G.E (2009). Melarsoprol cyclodextrin inclusion complexes. Faculty of Veterinary Medicine, Division of Infection and Immunity Seminar Series. Jones, A., Rodgers, J., Bradley, B., Kennedy, P.G.E (2009). Novel approaches to chemotherapy in a murine model of human African trypanosomiasis. Faculty of Veterinary Medicine Annual Postgraduate Pfizer Prize. IX