Pharmacology of DB844, an Orally Active aza Analogue of Pafuramidine, in a Monkey Model of Second Stage Human African Trypanosomiasis

Novel drugs to treat human African trypanosomiasis (HAT) are still urgently needed despite the recent addition of nifurtimox-eflornithine combination therapy (NECT) to WHO Model Lists of Essential Medicines against second stage HAT, where parasites have invaded the central nervous system (CNS). The pharmacology of a potential orally available lead compound, N-methoxy-6-{5-[4-(N-methoxyamidino) phenyl]-furan-2-yl}-nicotinamidine (DB844), was evaluated in a vervet monkey model of second stage HAT, following promising results in mice. DB844 was administered orally to vervet monkeys, beginning 28 days post infection (DPI) with Trypanosoma brucei rhodesiense KETRI 2537. DB844 was absorbed and converted to the active metabolite 6-[5-(4-phenylamidinophenyl)-furanyl-2-y​l]-nicotinamide(DB820), exhibiting plasma Cmax values of 430 and 190 nM for DB844 and DB820, respectively, after the 14th dose at 6 mg/kg qd. A 100-fold reduction in blood trypanosome counts was observed within 24 h of the third dose and, at the end of treatment evaluation performed four days post the last drug dose, trypanosomes were not detected in the blood or cerebrospinal fluid of any monkey. However, some animals relapsed during the 300 days of post treatment monitoring, resulting in a cure rate of 3/8 (37.5%) and 3/7 (42.9%) for the 5 mg/kg×10 days and the 6 mg/kg×14 days dose regimens respectively. These DB844 efficacy data were an improvement compared with pentamidine and pafuramidine both of which were previously shown to be non-curative in this model of CNS stage HAT. These data show that synthesis of novel diamidines with improved activity against CNS-stage HAT was possible.This investigation received financial support from the Bill and Melinda Gates Foundation through the Consortium for Parasitic Drug Development

Changes in red cell distribution width in monkeys following infection and subsequent treatment with DB844.

<p>Symbols and error bars represent means and SEs, respectively, of seven monkeys that were treated with DB844 at 6 mg/kg×14 days, from 28–41 days post infection.</p

Haematologic effects of <i>T. b. rhodesiense</i> KETRI2537 infection and treatment with DB844 in vervet monkeys.

<p>Key: RBC = red blood cells; WBC = White blood cells; g/dl = grams/decilitre; fl = femtolitres; p-values<0.05 indicate values that were significantly different from pre-infection baseline (day 0) values (Repeated measures Anova with Fishers PLSD post hoc test); Monkey were treated with DB844 at 6 mg/kg×14 days, from 28–41 days post infection.</p

HPLC/UV chromatograms and concentration-time profiles of DB844/metabolites following incubation of DB844 with male vervet monkey liver microsomes.

<p>A: HPLC/UV chromatograms; B: Concentration-time profiles of DB844 and metabolites. Incubation mixtures (1 ml at pH7.4, in triplicate) contained 10 µM DB844 and 0.2 mg/ml monkey liver microsomes. Aliquots were taken at 0.2, 5, 15, 30, and 120 min and evaluated for concentrations of DB844 and six metabolites (M1A, M1B, M2A, M2B, M3, and DB820). Metabolites M4A and M4B were not quantified due to lack of synthetic standards.</p

Plasma concentration-time profiles following oral administration of the last (14^th) daily dose of DB844.

<p>Symbols and error bars represent geometric means and SEs, respectively, for DB844 (△) and DB820 (○). The monkeys (n = 7) were treated with DB844 at 6 mg/kg×14 days, from 28–41 days post infection. The insert graph shows the extended profile up to 28 days post the last daily dose of DB844.</p

Treatment outcome in monkeys treated with DB844 while in second stage <i>T. b. rhodesiense</i> infection.

<p>Key: ID = identity in the laboratory; PP = pre-patent period; DPI = days post infection; CSF = cerebrospinal fluid; Tryps = trypanosomes; WC = white cells; EoT = end of treatment; ToC = test of cure; Neg = Negative; Pos = positive; WD = withdrawn from the experiment after 10^th drug dose due to toxicity; Numbers in square brackets = maximum number of white cell counts observed during any of the four weekly samplings between 0–27 DPI; I: DB844 5 mg/kg×10 days per os; 28–37 DPI; II: DB844 6 mg/kg×14 days per os; 28–41 DPI.</p

Parasitaemia pattern in monkeys infected with <i>T.b. rhodesiense</i> KETRI 2537 and subsequently treated with DB844.

<p>Symbols and error bars represent means and SEs, respectively, of 7 animals; monkeys were treated with DB844 at 6 mg/kg×14 days, from 28–41 days post infection; Log parasitaemia values were determined by microscopic examination of wet smears of blood using the matching method of Herbert and Lumsden, 1976 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001734#pntd.0001734-Ndungu1" target="_blank">[23]</a>.</p

Chemical structure of DB844 and DB820.

<p>Chemical structure of DB844 and DB820.</p

Transient infection and DB844 induced changes in clinical chemistry indicators of liver and kidney function.

<p>Symbols represent mean ± SE (n = 7) of aspartate aminotransferase (AST, ▪), alanine aminotransferase (ALT, □), total bilirubin (•), direct bilirubin (0), blood urea nitrogen (BUN, ▴) and albumin (◊); monkeys were treated with DB844 at 6 mg/kg×14 days, from 28–41 days post infection.</p