Jones, Amy (2011) Melarsoprol cyclodextrin inclusion complexes for the treatment for human ...

322 Pages·2011·6.55 MB·English

Jones, Amy (2011) Melarsoprol cyclodextrin inclusion complexes for the
treatment for human African trypanosomiasis. PhD thesis.

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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.
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