Melarsoprol cyclodextrin inclusion complexes for the treatment for human African trypanosomiasis

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

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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 dependant on the trivalent arsenical melarsoprol. Melarsoprol is an extremely toxic compound, the administration of which is very painful and associated with numerous adverse reactions. The most series of which is a post treatment reactive encephalopy (PTRE). The PTRE occurs in up to 10% of all patients given melarsoprol of which 50% die as a result of the complication. This gives melarsoprol an overall fatality rate of 5% which is unacceptably high. There is therefore an urgent need for new trypanocides, which are safe and easily administrable.
To improve the physiochemical and pharmacokinetic properties of melarsoprol the drug was complexed with two cyclodextrin molecules, hydroxypropyl-cyclodextrin (HPCD) and randomly methylated-cyclodextrin (RAMCD) to produce; mel/HPCD and mel/RAMCD. 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. The trypanocidal activity of melarsoprol is retained following its complexation with HPCD and RAMCD. The in-vitro trypanocidal activity of the melarsoprol cyclodextrin complexes against bloodstream T. b. 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/HPCD and mel/RAMCD produced 100% cure rates when administered orally at a dose of 0.05mmol/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 HPCD and RAMCD 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/HPCD and mel/RAMCD 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/HPCD and five doses of mel/RAMCD. The elimination of all trypanosomes from the CNS following four doses of mel/HPCD and five doses of mel/RAMCD, indicates that it may be possible to reduce the dosage schedule from seven daily doses to four daily doses of mel/HPCD and five doses of mel/RAMCD. 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 curative mel/HPCD treatment on the BBB was investigated in a murine model of CNS T. b. brucei infection using small bore MRI analysis. Mel/HPCD treatment results in a rapid restoration of BBB function as by twenty-four hours after the completion of mel/HPCD therapy the integrity of the BBB was fully restored. However, a very 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/HPCD and mel/RAMCD. 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/HPCD was below the limit of quantification of the assay. The pharmacokinetics of mel/HPCD and mel/RAMCD could therefore not be determined in the present study.
This study demonstrates that the melarsoprol cyclodextrin complexes mel/HPCD and mel/RAMCD are highly trypanocidal with no overt signs of toxicity and more importantly orally available. Following the oral administration of mel/HPCD or mel/RAMCD 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/HPCD and mel/RAMCD are promising oral candidates for the treatment of HAT.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Human African trypanosomiasis, melarsoprol, cyclodextrin
Subjects: S Agriculture > SF Animal culture > SF600 Veterinary Medicine
Q Science > QL Zoology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Biodiversity, One Health & Veterinary Medicine
Supervisor's Name: Jean, Dr. Rodgers
Date of Award: 2011
Depositing User: Miss Amy Jones
Unique ID: glathesis:2011-2713
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 20 Jun 2011
Last Modified: 26 Jun 2014 13:42

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