Discovery of novel active diamidines as clinical candidates against "Trypanosoma evansi" infection

The animal pathogenic protozoan, Trypanosoma evansi, leads to a wasting disease in equines, cattle and camels, commonly known as Surra. It is extensively distributed geographically with a wide range of mammalian hosts and causes great economical loss, especially in areas of Africa, Asia and South America. Transmitted mechanically by biting flies (tabanids), the most effective form of control for this disease appears to be chemotherapy and treatment is currently based on four main drugs: suramin, diminazene aceturate, quinapyramine and cymelarsan. However, due to emerging resistance to these drugs, their toxicity and a sparse drug repertoire, their effective use is severely threatened, emphasising the urgent necessity in finding new alternative drugs.
Diamidines are dicationic molecules, which bind to the minor groove of DNA at AT-rich sites and then either inhibit one or more DNA dependent enzymes, or directly impede the transcription process. It has been known since the 1940s that these molecules contain anti-protozoal properties. The diamidine drug, pentamidine, is currently used to treat Pneumocystis carinii pneumonia, leishmaniasis and the early stage of African trypanosomiasis. Yet pentamidine is impeded by its toxicity, its inability to be absorbed orally and a limited understanding of its mode of action. New diamidine compounds were recently synthesised, revealing excellent in vitro and in vivo activity against human African trypanosomiasis.
The overall aim of this project was to explore the possibility of finding new diamidines with considerable activity against Trypanosoma evansi and to identify any novel diamidine compounds, which possess the potential to act as clinical candidates against this infection. Investigations carried out in this thesis using Trypanosoma evansi demonstrated outstanding in vitro activities for 181 diamidines, with IC50 values below 30 ng/ml. These compounds were further examined within an in vivo mouse model to determine the lowest curative dose and to ascertain preliminary toxicity values of the most active diamidines. A total of 49 compounds were examined for their in vivo efficacy, of which six were capable of curing Trypanosoma evansi infected mice at 0.5 mg/kg doses, given over four consecutive days. Additionally, a series of Trypanosoma evansi strains, isolated from various geographical locations, were also tested, to establish the effective sensitivity of such chemicals to different strains.
Based on these in vitro and in vivo findings, three novel diamidines (DB 75, DB 867 and DB 1192) were selected and tested for preliminary acute toxicity within a goat model. Several toxicity trials were performed, whereby a single, 4mg/kg dose of each compound was given to goats and a multiple dose was given as four 1 mg/kg drug applications over six hours. All drug injections were given to the goats via an intramuscular route of application. As well as collecting pharmacokinetic data related to the toxicity trials, the goats were additionally observed for any signs of acute toxicity, of which none were seen at a total dose of 4 mg/kg for any of the compounds tested. Thereafter, tissue and organ samples were collected from each goat and prepared for histological examination.
Having concluded that DB 75, DB 867 and DB 1192 were considered safe in goats, they were furthermore investigated within an experimentally infected Trypanosoma evansi goat model. The efficacy of these compounds was examined at doses of 2.5 mg/kg and 1.25 mg/kg. Diminazene was included as a positive control drug and was given as 5 mg/kg. After treatment of the infected goats, a five month follow up study was conducted to determine the curative potential of the compounds used. Curative efficacy was assessed using three types of detection tests; a parasitological test (Haematocrit Centrifugation Technique), a serological test (CATT / T. evansi) and a molecular based test (PCR / T. evansi). At the end of the follow up efficacy trial, it was seen that DB 75 was able to effectively cure infected goats at a quarter of the dose (1.25 mg/kg) used by the positive control drug (Diminazene) and that DB 867 could cure infected goats at half the dose (2.5 mg/kg) used by that of diminazene. The third compound, DB 1192 was not able to treat goats at either the high or low doses tested and the infected goats relapsed, just two weeks post treatment.
Moreover, pharmacokinetic data was gathered from the efficacy study and the plasma samples collected were analysed using HPLC/MS-MS techniques, to determine the drug concentrations present in the goats over several time points. This enabled distinct pharmacokinetic profiles to be produced, revealing a clearer comprehension as to why DB 1192 failed to treat the experimentally infected goats. Drug concentrations obtained in plasma samples for DB 75 and DB 867 were found to be well within the therapeutic range. Several pharmacokinetic parameters were calculated to provide a better understanding of diamidines within goats. Furthermore, the long half lives of DB 75 and DB 867 may indicate drug accumulation within tissues, whilst the kinetoplast, nucleus and acidocalcisomes were identified as structures within trypanosomes where these diamidines accumulate, based on preliminary fluorescent experiments.
In conclusion, two novel diamidine compounds (DB 75 and DB 867) have been identified as potential clinical candidates against Trypanosoma evansi infection.