Rasmus Henninger1, Katrien Van Bocxlaer2, Wietse M. Schouten3, Charles E. Mowbray4, Jadel Müller Kratz5, Thomas P. C. Dorlo1
1Department of Pharmacy, Uppsala University, 2Skin Research Centre, Hull York Medical School, York Biomedical Research Institute, University of York, 3Department of Pharmacy & Pharmacology, Antoni van Leeuwenhoek/The Netherlands Cancer Institute, 4Drugs for Neglected Diseases initiative, DNDi, 5Drugs for Neglected Diseases initiative, DNDi
Introduction: Cutaneous leishmaniasis (CL), the most common form of leishmaniasis, is a neglected tropical disease caused by Leishmania (L.) parasites [1]. In 2023, over 270,000 new cases were reported, primarily affecting impoverished populations [2]. During a blood meal, sand flies – the vectors of Leishmania – inject Leishmania parasites into the subcutaneous tissue, where they are phagocytosed by immune cells and begin to replicate [3]. CL manifests as skin ulcers that may resolve spontaneously or develop into chronic, disfiguring lesions. Traditional preclinical PD markers for CL include microscopic analysis of skin biopsies and parasite quantification from tissue homogenates using limiting dilution assays or qPCR. However, these methods provide only a static assessment of infection, as target skin tissue collection typically requires animal sacrifice. In vivo bioluminescent imaging offers a less invasive and dynamic alternative for evaluating drug candidate efficacy using luciferase-expressing parasites [4]. Objectives: This study aimed to characterize the relationship between the skin target site PK of DNDI-6148, parasite clearance as quantified by bioluminescent imaging from luciferase-expressing L. major promastigotes, and lesion healing dynamics in mice. Methods: PD data originated from a dose-response study where 24 female BALB/c mice were infected with L. major Friedlin promastigotes in the rump. Once lesions and bioluminescence reached a defined threshold, mice were grouped based on similar bioluminescence signals (n=6). DNDI-6148 was administered via oral gavage at four dosage levels (6.25, 12.5, 25, and 50 mg/kg) for 10 consecutive days. Bioluminescence in mouse skin was quantified using IVIS imaging after subcutaneous d-luciferin injection, anaesthesia, and signal acquisition with exposure times adjusted for intensity. Background bioluminescence was subtracted from parasite signals, while lesion size was measured daily and converted to area using the ellipse formula. The PKPD relationships were characterized with an NLME approach in NONMEM 7.5, incorporating skin drug exposure from a previously developed plasma/skin PK model [5]. Exposure targets for 95% and 99% parasite reduction in the skin were identified by simulating 10,000 murine PK/PD profiles, determining the doses needed for 95% of the population to achieve these reductions. Results: A total of 165 parasite bioluminescence and 237 lesion size measurements were collected from 24 mice treated with DNDI-6148. Parasite burden was described by a constant baseline, with an estimated typical value of 8.01 log10 photons/s [7.91–8.10], and an associated high BSV of 60% CV, assuming no net growth and suggesting the parasite burden had reached a steady-state at the start of treatment. Drug-induced parasite clearance was best characterized by a sigmoidal Emax model, directly linked to the free skin target site concentration of DNDI-6148 over time, with the effect proportional to the skin drug concentration and no detectable delay. The maximal rate of parasite elimination (Emax) was estimated at 0.0490 hr-1 [0.0464–0.0516], with a half-maximal effect (EC50) achieved at DNDI-6148 unbound skin concentration of 165 µg/L [121–208]. Higher parasite load was correlated with increased lesion size. Therefore, the lesion-parasite relationship was modelled with a dynamic lesion growth rate (kles) proportional to parasite load over time and a constant lesion healing rate (kheal). Due to the high parasite load at baseline, kles was calculated as 0.0232 hr?¹ on day 0. By the end of treatment, kles was negligible due to drug-induced parasite clearance, while lesion healing was primarily driven by kheal, estimated at 0.0272 hr?¹ [0.0102–0.0442], resulting in a typical skin turnover time (1/k) of 1.5 days. Probability of target attainment analysis indicated that DNDI-6148 BID dosing of 17.5 mg/kg and 25 mg/kg over 10 days would achieve 95% and 99% parasite reduction in >95% of mice. Conclusions: The developed PKPD model successfully described the relationship between DNDI-6148 concentrations at the target skin site of infection and rate of parasite kill by in vivo bioluminescence imaging in a murine L. major model. The doses that resulted in 95% and 99% parasite reduction were 17.5 and 25 mg/kg, respectively. The established exposure-response relationship, adjusted for differences in unbound fraction, will be extrapolated across species and integrated with clinical PK to predict a human efficacious dose of DNDI-6148 for the treatment of CL.
[1] Leishmaniasis (2025). Available from: https://www.who.int/news-room/fact-sheets/detail/leishmaniasis [2] Global leishmaniasis surveillance updates 2023: 3 years of the NTD road map. WHO Reference Number: WER No. 45, 2024 99, 653–669. Available from: https://www.who.int/publications/i/item/who-wer-9945-653-669 [3] Scott, P., Novais, F. Cutaneous leishmaniasis: immune responses in protection and pathogenesis. Nat Rev Immunol 16, 581–592 (2016). https://doi.org/10.1038/nri.2016.72 [4] Mendes Costa, D., Cecílio, P., Santarém, N. et al. Murine infection with bioluminescent Leishmania infantum axenic amastigotes applied to drug discovery. Sci Rep 9, 18989 (2019). https://doi.org/10.1038/s41598-019-55474-3 [5] Henninger R et al PAGE 32 (2024) Abstr 11192 [www.page-meeting.org/?abstract=11192]
Reference: PAGE 33 (2025) Abstr 11469 [www.page-meeting.org/?abstract=11469]
Poster: Drug/Disease Modelling - Infection