Summary: In this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb‐S; and antimony resistant Sb‐R). MIL‐R was easily induced in both strains using the promastigote‐stage, but a significant increase in MIL‐R in the intracellular amastigote compared to the corresponding wild‐type did not occur until promastigotes had adapted to 12.2 μM MIL. A variety of common and strain‐specific genetic changes were discovered in MIL‐adapted parasites, including deletions at the LdMT transporter gene, single‐base mutations and changes in somy. The most obvious lipid changes in MIL‐R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL‐R parasites, with more genetic changes occurring in Sb‐R compared with Sb‐S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb‐R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite's biochemical pathways and how they are genetically regulated to be verified fully.

The antileishmanial efficacies of four proprietary amphotericin B (AmB) formulations (Fungizone, AmBisome, Abelcet, and Amphocil) and an experimental nonionic surfactant vesicle (NIV) formulation were compared in a murine model of acute visceral leishmaniasis. By a multiple-dosing regimen, groups of Leishmania donovani-infected BALB/c mice were treated (2.5 mg of AmB per kg of body weight) on days 7 to 11 postinfection with one of the AmB formulations, and parasite burdens were determined on day 18 postinfection. All of the formulations caused significant suppression parasite burdens in spleens (P < 0.01 to 0.0005) and livers (P < 0.0005) compared with those in the spleens and livers of the controls. In addition, a significant suppression of parasite burdens in bone marrow (P < 0.0005) compared to the burdens in the bone marrow of the controls was obtained for all the formulations except Abelcet, which was inactive at this site. On the basis of their overall efficacies (activity against liver, spleen, and bone marrow parasites), the formulations could be ranked as follows: Amphocil = AmBisome > AmB-NIV > Abelcet > Fungizone. On the basis of spectrophotometric measurements, AmB was shown to exist in a predominantly aggregated state in all of the formulations. Although incubation in 50% serum altered the degree of aggregation, the AmB remained predominantly aggregated, indicating that the AMB-lipid complex in all of the formulations was physically stable. The results of the study showed that antiparasitic efficacy is associated positively with the degree of AmB aggregation in the presence of serum.

Resistance to pentavalent antimonial (Sb ^v ) agents such as sodium stibogluconate (SSG) is creating a major problem in the treatment of visceral leishmaniasis. In the present study the in vivo susceptibilities of Leishmania donovani strains, typed as SSG resistant (strain 200011) or SSG sensitive (strain 200016) on the basis of their responses to a single SSG dose of 300 mg of Sb ^v /kg of body weight, to other antileishmanial drugs were determined. In addition, the role of glutathione in SSG resistance was investigated by determining the influence on SSG treatment of concomitant treatment with a nonionic surfactant vesicle formulation of buthionine sulfoximine (BSO), a specific inhibitor of the enzyme γ-glutamylcysteine synthetase which is involved in glutathione biosynthesis, and SSG, on the efficacy of SSG treatment. L. donovani strains that were SSG resistant (strain 200011) and SSG sensitive (strain 200016) were equally susceptible to in vivo treatment with miltefosine, paromomycin and amphotericin B (Fungizone and AmBisome) formulations. Combined treatment with SSG and vesicular BSO significantly increased the in vivo efficacy of SSG against both the 200011 and the 200016 L. donovani strains. However, joint treatment that included high SSG doses was unexpectedly associated with toxicity. Measurement of glutathione levels in the spleens and livers of treated mice showed that the ability of the combined therapy to inhibit glutathione levels was also dependent on the SSG dose used and that the combined treatment exhibited organ-dependent effects. The SSG resistance exhibited by the L. donovani strains was not associated with cross-resistance to other classes of compounds and could be reversed by treatment with an inhibitor of glutathione biosynthesis, indicating that clinical resistance to antimonial drugs should not affect the antileishmanial efficacies of alternative drugs. In addition, it should be possible to identify a treatment regimen that could reverse antimony resistance.

In this study, treatment efficacies of a nonionic surfactant vesicle formulation of sodium stibogluconate (SSG-NIV) and of several formulations of amphotericin B were compared in a murine model of visceral leishmaniasis. Treatment with multiple doses of AmBisome, Abelcet, and Amphocil (total dose, 12.5 mg of amphotericin B/kg of body weight) resulted in a significant suppression of parasite burdens in liver ( P < 0.0005) and spleen ( P < 0.0005) compared with those of controls, with Abelcet having the lowest activity. Only AmBisome and Amphocil gave significant suppression of parasites in bone marrow (compared to control values, P < 0.005). In the acute-infection model, single-dose treatments of SSG-NIV (296 mg of Sb ^V /kg), SSG solution (296 mg of Sb ^V /kg), or AmBisome (8 mg of amphotericin B/kg) were equally effective against liver parasites (compared to control values, P < 0.0005). SSG-NIV and AmBisome treatment also significantly suppressed parasites in bone marrow and spleen ( P < 0.005), with SSG-NIV treatment being more suppressive (>98% suppression in all three sites). Free-SSG treatment failed to suppress spleen or bone marrow parasites. Infection status influenced treatment outcome. In the chronic-infection model, the AmBisome single-dose treatment was less effective in all three infection sites and the SSG-NIV single-dose treatment was less effective in the spleen. The results of this study suggest that the antileishmanial efficacy of SSG-NIV compares favorably with those of the novel amphotericin B formulations.

Sequencing studies showed that the γ-glutamylcysteine synthetase (γ-GCS) heavy chain genes from sodium stibogluconate (SSG)-resistant (SSG-R) and SSG-susceptible (SSG-S)Leishmania donovanistrains were identical, indicating that SSG resistance was related to quantitative differences in γ-GCS expression rather than gene interstrain polymorphisms. In vitro infection of murine macrophages with the SSG-R strain, but not the SSG-S strain, down regulated expression of host γ-GCS, which would result in a reduction in intramacrophage glutathione (GSH) levels and promote an oxidative intramacrophage environment. This would inhibit, or minimize, the reduction of SSG pentavalent antimony to its more toxic trivalent form. Macrophage studies showed that the SSG-R strain expressed higher levels of γ-GCS compared to the SSG-S strain, which would result in higher GSH levels, giving increased protection against oxidative stress and facilitating SSG efflux. However a similar differential effect on host and parasite γ-GCS expression was not obtained when using tissues from infected mice. In this case γ-GCS expression was organ and strain dependent for both the host and the parasite, indicating that environmental conditions have a profound effect on γ-GCS expression. Consistent with the proposed mechanism from in vitro studies, increasing tissue GSH levels in the presence of SSG by cotreatment ofL. donovani-infected mice with SSG solution and GSH incorporated into nonionic surfactant vesicles was more effective in reducing liver, spleen, and bone marrow parasite burdens than monotherapy with SSG. Together, these results indicate that SSG resistance is associated with manipulation of both host and parasite GSH levels byL. donovani.

Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMM ^r ) was induced in three Nepalese clinical strains of Leishmania donovani with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM.