Cinnamic Acid Bornyl Ester Derivatives from <i>Valeriana wallichii</i> Exhibit Antileishmanial <i>In Vivo</i> Activity in <i>Leishmania major</i>-Infected BALB/c Mice

<div><p>Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by <i>Leishmania major</i> or <i>Leishmania donovani</i>, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (<b>1</b> and <b>2</b>) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (<b>2</b>) <i>in vitro</i> prompted the antileishmanial assessment <i>in vivo</i>. For this purpose, BALB/c mice were infected with <i>Leishmania major</i> promastigotes and treated with three doses of 50 mg/kg/day of compound <b>2</b>. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of <i>Leishmania major</i> promastigotes revealed that compounds <b>1</b> and <b>2</b> induce mitochondrial swelling. Subsequent studies on <i>Leishmania major</i> promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨ_m) as a putative mode of action. As the cinnamic acid bornyl ester derivatives <b>1</b> and <b>2</b> had exhibited antileishmanial activity <i>in vitro</i>, and compound <b>2</b> in <i>Leishmania major</i>-infected BALB/c mice <i>in vivo</i>, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches.</p></div

Compound-associated cell death is time-dependent.

<p><i>L</i>. <i>major</i> promastigotes were treated with solvent control (1% DMSO), apoptosis inducer miltefosine (122.7 μM), compound 1 (100 μM), and 2 (100 μM) in a time course experiment for 6 h, 10 h, and 24 h. Cells were harvested, stained with AV and PI and subsequently analyzed by flow cytometry. Analysis for compound 2 was performed in an independent experiment. Percentage for each quadrant is written below the corresponding dot blot figure. Lower-left (LL): live cells (AV^-/PI^-); upper-left (UL): early necrotic cells (AV^-/PI⁺); upper-right (UR): necrotic/late apoptotic cells (AV⁺/PI⁺); and lower-right (LR): early apoptotic cells (AV⁺/PI^-).</p

Compound 2 treatment of <i>L</i>. <i>major</i>-infected BALB/c mice is associated with reduced parasite burden in infected tissues and organs.

<p>Female BALB/c mice (n = 33) were infected with 5 × 10⁶<i>L</i>. <i>major</i> promastigotes into the right hind footpad. Treatment regimen started on d 21 post infection either using 50% DMSO/PBS (□) (n = 11), 50 mg/kg compound 2 (■) (n = 11) or mice were left untreated (▲) (n = 11). A. At the end of experiment on d 35 all mice (n = 33) were sacrificed and the parasite burden of the infected footpads (iFP) (n = 9) was determined using Limited Dilution Assays (LDA). B. Photographic documentation of the infected footpads of three exemplary mice per group shall reveal the severity of disease progression in non-healing mice and the control of clinical manifestation in compound 2-treated BALB/c mice. C. The parasite burden of the infection site-draining popliteal lymph nodes (pLN) of individual mice (n = 9) or D. spleens of individual mice (n = 6) was determined using LDA. n = number of animals; ** p<0.005; differences in parasite load between 50% DMSO/PBS-treated and compound 2-treated animals did not reach statistical significance for D.</p

The tested compounds 1 and 2 affect mitochondrial integrity and membrane potential.

<p>Mitochondrial integrity of compound-treated <i>L</i>. <i>major</i> promastigotes was investigated using flowcytometric determination of MitoTracker Red CMXRos accumulation within active organelles. <i>L</i>. <i>major</i> promastigotes were incubated in 1% DMSO or treated with 100 μM compound 1 or 2 for indicated time points prior to MitoTracker Red CMXRos staining. MitoTracker-positive cell populations were determined by histograms analysis and were presented as percentage of control cells (set as 100%, inserted line).</p

Determination of cytotoxicity and IC₅₀ values against <i>Leishmania</i> parasites.

<p>Determination of cytotoxicity and IC₅₀ values against <i>Leishmania</i> parasites.</p

Changes in cell morphology in <i>L</i>. <i>major</i> promastigotes upon treatment.

<p>Compound 1 and 2 induce parasite swelling and vacuolization of <i>L</i>. <i>major</i> promastigotes at very early time points. <i>L</i>. <i>major</i> promastigotes were treated with solvent control (1% DMSO), miltefosine (122.7 μM), compound 1 (100 μM), and 2 (100 μM) for 30 min, 1 h, 2 h, 4 h, and 24 h. Cytospin preparations of the cells were stained with Diff-Quik dye and analyzed by transmitted light microscopy under 50× objective. “Plus” symbol (+) was used to represent changes in morphology; two (++) and three (+++) symbols were used as a reference for the severity of the phenotype induced by the different tested compounds and the black arrows to indicate the presence of vacuole-like structures. Black bar indicates 20 μm.</p