Route map for the discovery and pre-clinical development of new drugs and treatments for cutaneous leishmaniasis

Abstract

Although there have been significant advances in the treatment of visceral leishmaniasis (VL) and several novel compounds are currently in pre-clinical and clinical development for this manifestation of leishmaniasis, there have been limited advances in drug research and development (R & D) for cutaneous leishmaniasis (CL). Here we review the need for new treatments for CL, describe in vitro and in vivo assays, models and approaches taken over the past decade to establish a pathway for the discovery, and pre-clinical development of new drugs for CL. These recent advances include novel mouse models of infection using bioluminescent Leishmania, the introduction of PK/PD approaches to skin infection, and defined pre-clinical candidate profiles.

Keywords: Cutaneous leishmaniasis; Drug development; Drug discovery; Formulations; Immunomodulatory drugs; In vitro assays; In vivo models; Pharmacokinetics.

Graphical abstract

Fig. 1

WRAIR Cutaneous Leishmaniasis Testing Strategy. Analogs, designed or acquired, are assessed for activity (intracellular amastigote) and potential cytotoxicity. All analogs meeting cutoff criteria (assigned per case) but with a therapeutic index > 5 are tested for microsomal stability in mouse and human microsomes. These higher throughput assays gate the more costly, clinically relevant, animal models. In vivo efficacy in mice is initially tested against Old World (L. major) parasites, first in a lesion suppression model, then if warranted advanced to the rigorous lesion cure model. Initial in vivo experiments use intraperitoneal (IP) route of administration to maximize chance of success and provide early dose ranging. If successful, skin and plasma mouse PK is performed followed by oral (PO) dosing in the mouse lesion cure model (supporting our TPP). The final tier of in vivo efficacy assesses activity via PO dosing in a second animal species (GSH) as required for FDA approval and efficacy against a New World strain of Leishmaniasis in a mouse footpad model. Once late-stage candidates are identified early safety, assessment is performed as outlined above.

Fig. 2

Disseminated Leishmania disease in BALB/c Mice. Mice were infected at the base of the tail with 1 × 10⁷ stationary phase luciferase expressing L. major parasites and the images were obtained through three dimensional reconstruction of bioluminescence using DLIT in a BALB/c mouse at forty days (image A) and seventy days (image B) post-infection. The brighter, red and yellow areas shown in the image are photon intensity measurements representing greater parasite counts than the dimmer green-blue areas. Forty days post infections (image A) the L. major parasites are visible only in the infected skin at edge of the infection (lesion) site*. At seventy days post infections (image B) the L. major parasites are visible in large quantities in both popliteal and axillary lymph nodes (PLN, ALN) and possibly in the internal organs.* In our laboratory, using the in vivo imaging technology, the smallest number of parasites that can be visualized in BALB/c mice's skin immediately after infection is 1.5x10^∧4 luciferase-expressing L. major promastigotes. The limit of detection for amastigotes in the skin and internal organs has not been determined. Other methods can detect presence of parasites in the internal organs at a much earlier time during the disease progression. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

A Drug discovery progression pathway for CL including the evaluation of both pharmacokinetic and pharmacodynamic parameters.

Fig. 4

A schematic representation of the skin at different stages of CL. An early form of CL is shown in (A) where the skin is visibly still intact but a small nodule or papule is visible; (B) shows the more advanced stage whereby the crust was removed and an ulcer is exposed. The epidermal layers of the skin (including the stratum corneum) are absent. As part of the healing process, a crust is formed (C).

Fig. 5

A schematic representation of a permeation assay using a 24-well plate system (A) or a Franz diffusion cell (B).