Pharmacological investigations on current and new drugs for treatment of human African trypanosomiasis

Trypanosomiasis remains a major threat to humans in sub-Saharan Africa with 55-60 million people in 36 countries at risk of infection with T.b. gambiense or T.b. rhodesiense. Only a few drugs are available to treat the disease, which are either toxic, scarce, or do not readily pass the blood brain barrier to achieve therapeutic levels in the brain. A new drug is not in sight. The organo-arsenical compound melarsoprol is the main drug in use for therapy of the late stage of the disease, with central nervous system involvement. During melarsoprol treatment patients often suffer from serious adverse effects such as fever, diarrhea, pruritus and in 2-10% of the cases from reactive encephalopathy with fatal outcome in 50%. Although melarsoprol was introduced in 1949 there is a lack of data on its metabolites, pharmacologic or chemical properties. The present PhD project had two objectives: Firstly to analyze, characterize and determine the pharmacokinetics of the metabolites of the trypanocidal drug melarsoprol (the pharmacokinetics of relapse patients were additionally investigated). Secondly to evaluate further possible drug candidates for treatment of trypanosomiasis by structure activity relationship: In this study a group of antibacterial agents, the fluoroquinolones, was examined. A HPLC-UV method was developed to separate the parent drug melarsoprol from its potential metabolites. One metabolite could be identified after incubation of the drug with microsomes and also in serum of patients treated with melarsoprol: Melarsen oxide. Interestingly, this trypanocidal drug was in use before the development of melarsoprol, which is the condensation product of melarsen oxide and dimercaprol (an antidote for arsenic poisoning). After administration of 2.2 mg/kg of melarsoprol Cmax of melarsen oxide was reached after 5-15 minutes, the clearance was found to be 21.5 ml/min/kg and a half life of 3.8 hours was calculated. The therapeutic value of melarsen oxide was investigated in in vivo mice experiments: It could be demonstrated that the drug passes the blood brain barrier, hence might be an alternative to melarsoprol. Investigations on additional metabolites revealed that melarsen oxide or its arsenical derivatives are irreversibly bound to proteins. Covalent protein bound drugs are often associated with serious immunological adverse effects. The bacteriocidal class of fluoroquinolones act via the inhibition of topoisomerases. These enzymes mediating topological changes in the DNA are essential for cell survival. Trypanosomes possess a condensed, topologically interlocked circular type of mitochondrial DNA. The high demand for topoisomerase activity in combination with the rapid proliferation of trypanosomes makes the inhibition of topoisomerases an interesting drug target. 160 fluoroquinolones were tested against T.b. rhodesiense in vitro, with several compounds found to be active in the nanomolar range. The R7 substitution of the quinolone core was of importance for activity, whereas structural modification of the quinolone structure at position R1, R2, R3, and R8 did not influence trypanocidal activity. In vitro cytotoxicity was determined for active compounds and subsequently
six compounds were selected for further in vivo evaluation. However, even
after the administration of high doses (100 mg/kg) no parasitological cure of the mice
could be achieved.