Target site bioanalysis and pharmacokinetics to optimize antiparasitic drug development

Neglected vector-borne diseases, including malaria and cutaneous leishmaniasis, continue to cause a substantial global health burden, disproportionately affecting populations in low- and middle-income countries. Drug development for these parasitic diseases depends strongly on pharmacokinetics to guide compound selection, clinical translation, and dose optimization. Traditionally, systemic plasma pharmacokinetics has served as the primary basis for such decisions. However, plasma drug concentrations do not always reflect drug exposure at the actual site of infection, particularly for pathogens that reside in specific tissues or inside host cells. This has driven a shift toward target site pharmacokinetics, where drug concentrations are measured directly at the site of infection.

This thesis covered the development and validation of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methods for the quantification of antiparasitic drugs indicated for malaria or leishmaniasis. These included preclinical drug candidates from the Drugs for Neglected Diseases initiative (DNDi), for which animal studies inform first-in-human dose selection, and the approved antimalarial drug pyronaridine, for which clinical pharmacokinetic data support dose optimization. In these chapters, several recurring bioanalytical challenges, associated with target site pharmacokinetics, were addressed, including limited availability of biological matrices, incomplete tissue homogenization, and analyte instability during sample pre-treatment. To address the common practice of normalizing skin tissue drug concentrations to biopsy weight, a new workflow was also proposed that used the amount of double-stranded DNA in the biopsy, reducing variability caused by differences in tissue composition and sample handling.

The second part of the thesis describes the clinical pharmacokinetics of miltefosine, an antileishmanial drug used to treat cutaneous leishmaniasis, in vulnerable patient populations using minimally invasive sampling. This included the pharmacokinetics of miltefosine in pediatric patients with Old World cutaneous leishmaniasis in Afghanistan, and the systemic and skin pharmacokinetics of miltefosine in patients with cutaneous leishmaniasis in Ethiopia. As part of this work, a minimally invasive skin microsampling device was evaluated as a potential alternative to skin biopsies, but showed limited agreement with biopsy-based measurements, indicating that further work is needed to find sampling methods that reduce the burden on patients while still giving reliable exposure data. By assessing the pharmacokinetics of miltefosine in these clinical trials, dose optimization may help to improve clinical outcome in these patient populations.

Together, this thesis followed a specific line of work: bioanalytical methods that allow preclinical target site pharmacokinetic studies to inform first-in-human dosing, and that allow clinical pharmacokinetic studies to further optimize dosing in patients. By using minimally invasive sampling and better tissue-based quantification, this thesis aimed to lower the burden on patients while providing more clinically relevant exposure data. Taken together, this work showed that accurate bioanalytical methodology is a necessary basis for evidence-based drug development and dose optimization in malaria and cutaneous leishmaniasis, diseases that still receive limited research investment relative to their global impact.