Investigations on new and forgotten antischistosomal drugs and praziquantel pharmacokinetics in opisthorchiasis patients

Neglected tropical diseases (NTDs) are defined as a group of communicable diseases, most prevalent in tropical and subtropical regions of the world. Prevailing over the poorest populations that have limited access to sanitation, hygiene and education, NTDs affect more than one billion people worldwide and are endemic in 149 countries. Schistosomiasis, caused by blood-dwelling trematode flatworms of the Schistosoma genus is a neglected parasitic disease. The tremendous disease burden of schistosomiasis is associated with anemia, malnutrition as well as impaired childhood development and causes high morbidity for more than 200 million infected individuals. Praziquantel (PZQ) is the sole drug currently available; morbidity reduction using preventive chemotherapy relies completely on PZQ. Apart from its lack of activity against developing stages of the worm and the fact that PZQ is rarely curative, its abundant use incurs the possibility of drug resistance. Thus, there is an urgent need to identify and develop novel therapeutic principles. Opisthorchiasis, caused by the liver fluke Opisthorchis felineus is a food-borne trematodiasis with worldwide highest prevalence in Western Siberia. There are 12.5 million individuals at risk for an infection. The disease erupts upon dietary consumption of raw or undercooked fish infested with trematode larvae. It affects the hepatobiliary system and can lead to pancreatitis or even severe chronic infections such as hepatic abscesses. As for schistosomiasis, the drug of choice for the treatment of opisthorchiasis is praziquantel. The doses used are empirical and prescribed individually, the whole treatment scheme is very complicated and takes about three months to be accomplished; thus it makes it unavailable for many people. My PhD thesis had two major aims. The first was to advance antischistosomal drug discovery by identifying novel chemical entities and by rescuing a forgotten lead candidate. The second was to elucidate the pharmacokinetic properties of praziquantel in O. felineus infected patients, laying the groundwork for a simplified standardized treatment scheme. iv A first study we conducted to achieve the first aim; followed-up on an existing structure-activity relationship (SAR) program, the antischistosomal in vitro and in vivo activities of a 3rd generation pyridobenzimidazole (PBI) drug set were evaluated. We found high in vitro activities against Schistosoma mansoni newly transformed schistosomula (NTS) and adult worms, with IC50 values of 0.08 – 1.43 μM. Upon promising results obtained from metabolic stability assays with liver microsomes (>80%), we engaged in efficacy testing using infected mice. Moderate to high worm burden reductions (WBR) of 36 – 90 % were obtained after single oral doses of four selected compounds. However, we observed a small therapeutic window although results obtained from cytotoxicity screens against CHO and HepG2 cells did not foresee toxicity. In order to expand SAR studies around another promising chemotype, N,N’-diarylureas, we investigated a novel series of analogs featuring the pentafluorosulfanyl group (SF5). High in vitro activity, acceptable cytotoxicity profiles and high microsomal hepatic stability were found. IC50 values for the most promising compounds were between 0.8 and 1.8 μM for NTS and 0.1 – 0.4 μM for adult S. mansoni. However, none of the compounds tested in S. mansoni infected mice revealed in vivo efficacy. Results obtained from an accompanying PK study, showed that all tested compounds were slowly absorbed and only reached maximal plasma concentration values after 24 h. There was no correlation between drug exposure and in vivo efficacy. Extracts and fractions of the medicinal plants Artemisia annua and Artemisia afra were tested for their in vitro activity against S. mansoni NTS and adult worms. We comparatively evaluated a set of extracts and fractions and found low IC50 vales, especially for the A. afra (BB) hexane and DCM fractions. The findings suggest that A. afra, despite lacking the bioactive compound artemisinin found in A. annua, is more active than the latter, further warranting studies to identify novel molecules. Our studies on the largely forgotten lead candidate, Ro 15-5458, found high in vivo efficacy across the whole development stage of S. mansoni, when the drug was administered as a single oral dose of 50 mg/kg. Low ED50 of 5.3 and 15 mg/kg were obtained for mice harboring adult and juvenile infections, respectively. Ro 15-5458 was also tested against S. v haematobium and S. japonicum. We found an ED50 value of 17 mg/kg in hamsters harboring a patent S. haematobium infection; however, the compound was inactive at up to 100 mg/kg in S. japonicum infected mice. In vitro, a de-ethylated and a glucoronidated product were identified and the metabolic stability was evaluated using microsomes and hepatocytes. The low in vitro activity against NTS and adult worms and the late onset of action correlated with results obtained from hepatic shift experiments conducted S. mansoni infected mice. Ro 15- 5458 had a half-life of approximately 5 h and the maximal plasma concentration was reached after 3 h (8 µg/mL) when a 50 mg/kg oral dose was administered to healthy mice. In the context of a randomized controlled, single-blinded dose finding trial in Tomsk, Siberia, we conducted, for the second aim of this thesis, a PK study in O. felineus infected patients and assessed the kinetic disposition of both praziquantel enantiomers (R-PZQ and S-PZQ) and the drug’s main metabolite (R-trans-4-OH-PZQ). Single, ascending doses of 20, 40 and 60 mg/kg were compared to multiple dosing (3 x 60 mg/kg). Dried blood spots (DBS) collected over 24 h were analyzed using a validated LC-MS/MS method. Maximal plasma concentrations were reached after 1.5 h (1.72 μg/mL), 2 h (4.89 μg/mL) and 3 h (2.69 μg/mL) for R-, S- and R-trans-OH-PZQ, respectively, when praziquantel was given as a single 60 mg/kg dose. Highest exposure levels (AUC) were observed for the triple dose scheme with AUC vales of 8.04, 27.75 and 36.38 μg/mL*h, respectively. In conclusion, we present two sets of promising antischistosomal drug candidates, the PBIs and the N,N’-diarylureas. Our studies complement early drug discovery efforts around both chemotypes and provide insights for a next generation of optimized antischistosomal PBIs and N,N’-diarylureas. Additionally, extracts from medicinal plants such as A. annua and A. afra present potential alternatives to small molecules. Concurrently, Ro 15-5458 is an excellent lead candidate, efficacious alongside all stages of S. mansoni development, and further warrants investigations. Finally, praziquantel disposition in O. felineus infected individuals has been evaluated and in combination with the results from the efficacy study will be pivotal to establish an evidence based dosing recommendation