Treatment of African trypanosomosis with DB 75 : pharmacokinetics, relapses and cross resistance

Resurgent human African trypanosomosis (HAT) displays a major burden for people living in
Sub-Saharan Africa, although it had been nearly eradicated in the 50`s, due to selective
ground spraying with DDT and an extensive vector control. Today, treatment of HAT relies
only on a few drugs at hand. The use of these drugs is restricted, since they show an
unacceptable toxicity, complicated administration, emerging and spreading of resistance,
shortness of availability and/or show only an impact on gambiense or rhodesiense
trypanosomes. Therefore, new drugs are urgently needed.
A consortium, the UNC lead consortium to discover new drugs for the treatment of parasitic
diseases, was founded with the aim to improve treatment of HAT. The ideal profile of a new
drug is an uncomplicated synthesis, easy administration, high efficacy, cost effectiveness and
no toxicity. Series of compounds were synthesised in analogy to pentamidine, the first line
drug in the treatment of first stage trypanosomosis. The so called DB compounds showed
promising results in vitro and in vivo, not only to trypanosomes but also to Leishmania spp,
Mycobacterium tuberculosis, Plasmodium spp and Pneumocystis carinii. Lead compound of
this project is the diamidine DB 75 and its oral applicable prodrug DB 289.
Although the prodrug had entered an open label phase IIa clinical trial already when this
thesis started, several questions referring to the interplay between DB 75, trypanosomes and
mice had been unanswered. To elucidate development of parasitaemia in first infection, first
and second relapse in mice, studies have been undertaken in immunocompetent and –deficient
mice, with two different trypanosome strains and various treatment schedules for DB 75.
At the same time, parasite clearance after drug application was studied. Since relapse
trypanosomes did not show any differences in sensitivity to selected drugs, observed relapses
were not due to resistant trypanosomes but rather due to re-invasion from extravascular sites.
Identification of these sites was a second goal of this project. A method based on the detection
of trypanosomal mRNA by nested PCR was established and tested. Results showed that more
time has to be invested to increase the sensitivity of this method. However, one organ was
found positive for trypanosomal mRNA, indicating the spleen to be one of the niches.
A system was established to check DB 75 for cross resistance in melarsoprol and pentamidine
resistant laboratory strains. With the aid of this system, further upcoming drugs may be tested
for their cross resistance. For DB 75, only a low cross resistance was obtained in both
resistant strains.
Additionally, the pharmacokinetics for DB 75, DB 820 and the their corresponding prodrugs
DB 289, DB 844 in trypanosome infected and uninfected mice was studied. Differences in
plasma levels of uninfected compared to infected mice were obtained. Mainly, two effects
were shown to influence plasma levels considerably: accumulation of active drug in case of
DB 75 and DB 289, and inhibition of metabolism for DB 820 and DB 844.
The extent of DB 75 accumulation in trypanosomes was determined in vitro, to assess that the
differences between plasma levels of DB 75 after prodrug application obtained in infected
mice compared to uninfected mice were due to accumulation. In a further study, indications of
the presence of possible active metabolites were obtained when activity of plasma samples
was assessed by bioassay and compared to levels determined by HPLC/MS/MS.
In another pharmacokinetic study, it was shown that conversion of DB 289 to DB 75 in
P. berghei infected mice was poor, leading to subtherapeutic doses of active compound,
which explained that no activity in the in vivo model was obtained for the prodrug compared
to a moderate to good activity for the active compound.