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Activity of paromomycin against Leishmania amazonensis: Direct correlation between susceptibility in vitro and the treatment outcome in vivo.

ABSTRACT: Paromomycin is an aminoglycoside antibiotic approved in 2006 for the treatment of visceral leishmaniasis caused by Leishmania donovani in Southeast Asia. Although this drug is not approved for the treatment of visceral and cutaneous leishmaniasis in Brazil, it is urgent and necessary to evaluate the potential of this drug as alternative for the treatment against species responsible for these clinical forms of the disease. In Brazil, Leishmania amazonensis is responsible for cutaneous and diffuse cutaneous leishmaniasis. The diffuse cutaneous form of the disease is difficult to treat and frequent relapses are reported, mainly when the treatment is interrupted. Here, we evaluated paromomycin susceptibility in vitro of a L. amazonensis clinical isolate from a patient with cutaneous leishmaniasis and the reference strain L. amazonensis M2269, as well as its in vivo efficacy in a murine experimental model. Although never exposed to paromomycin, a significant differential susceptibility between these two lines was found. Paromomycin was highly active in vitro against the clinical isolate in both forms of the parasite, while its activity against the reference strain was less active. In vivo studies in mice infected with each one of these lines demonstrated that paromomycin reduces lesion size and parasite burden and a direct correlation between the susceptibility in vitro and the effectiveness of this drug in vivo was found. Our findings indicate that paromomycin efficacy in vivo is dependent on intrinsic susceptibility of the parasite. Beyond that, this study contributes for the evaluation of the potential use of paromomycin in chemotherapy of cutaneous leishmaniasis in Brazil caused by L. amazonensis.

SUBMITTER: Coser EM

PROVIDER: S-EPMC7548989 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Activity of paromomycin against Leishmania amazonensis: Direct correlation between susceptibility in vitro and the treatment outcome in vivo.

Coser Elizabeth M EM Ferreira Bianca A BA Branco Nilson N Yamashiro-Kanashiro Edite H EH Lindoso José Angelo L JAL Coelho Adriano C AC

International journal for parasitology. Drugs and drug resistance 20200815

Paromomycin is an aminoglycoside antibiotic approved in 2006 for the treatment of visceral leishmaniasis caused by Leishmania donovani in Southeast Asia. Although this drug is not approved for the treatment of visceral and cutaneous leishmaniasis in Brazil, it is urgent and necessary to evaluate the potential of this drug as alternative for the treatment against species responsible for these clinical forms of the disease. In Brazil, Leishmania amazonensis is responsible for cutaneous and diffuse ...[more]

PMID: 33011651

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Project description:Miltefosine was the first oral compound approved for visceral leishmaniasis chemotherapy, and its efficacy against Leishmania donovani has been well documented. Leishmania amazonensis is the second most prevalent species causing cutaneous leishmaniasis and the main etiological agent of diffuse cutaneous leishmaniasis in Brazil. Driven by the necessity of finding alternative therapeutic strategies for a chronic diffuse cutaneous leishmaniasis patient, we evaluated the susceptibility to miltefosine of the Leishmania amazonensis line isolated from this patient, who had not been previously treated with miltefosine. In vitro tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to miltefosine than L. amazonensis type strains. Due to this difference in susceptibility, we evaluated whether genes previously associated with miltefosine resistance were involved. No mutations were found in the miltefosine transporter gene or in the Ros3 or pyridoxal kinase genes. These analyses were conducted in parallel with the characterization of L. amazonensis mutant lines selected for miltefosine resistance using a conventional protocol to select resistance in vitro, i.e., exposure of promastigotes to increasing drug concentrations. In these mutant lines, a single nucleotide mutation G852E was found in the miltefosine transporter gene. In vivo studies were also performed to evaluate the correlation between in vitro susceptibility and in vivo efficacy. Miltefosine was effective in the treatment of BALB/c mice infected with the L. amazonensis type strain and with the diffuse cutaneous leishmaniasis isolate. On the other hand, animals infected with the resistant line bearing the mutated miltefosine transporter gene were completely refractory to miltefosine chemotherapy. These data highlight the difficulties in establishing correlations between in vitro susceptibility determinations and response to chemotherapy in vivo. This study contributed to establish that the miltefosine transporter is essential for drug activity in L. amazonensis and a potential molecular marker of miltefosine unresponsiveness in leishmaniasis patients.

Project description:Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05-500 μM and, when needed, 0.01-50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 μM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 μM for IA and 0.019 μM for IH). IA, IH and SMIH at 10 μM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results suggest that IA, IH and SMIH may provide new chemotherapy agents for leishmaniasis.

Project description:Nucleoside hydrolases of the Leishmania genus are vital enzymes for the replication of the DNA and conserved phylogenetic markers of the parasites. Leishmania donovani nucleoside hydrolase (NH36) induced a main CD4(+) T cell driven protective response against L. chagasi infection in mice which is directed against its C-terminal domain. In this study, we used the three recombinant domains of NH36: N-terminal domain (F1, amino acids 1-103), central domain (F2 aminoacids 104-198), and C-terminal domain (F3 amino acids 199-314) in combination with saponin and assayed their immunotherapeutic effect on Balb/c mice previously infected with L. amazonensis. We identified that the F1 and F3 peptides determined strong cross-immunotherapeutic effects, reducing the size of footpad lesions to 48 and 64%, and the parasite load in footpads to 82.6 and 81%, respectively. The F3 peptide induced the strongest anti-NH36 antibody response and intradermal response (IDR) against L. amazonenis and a high secretion of IFN-γ and TNF-α with reduced levels of IL-10. The F1 vaccine, induced similar increases of IgG2b antibodies and IFN-γ and TNF-α levels, but no IDR and no reduction of IL-10. The multiparameter flow cytometry analysis was used to assess the immune response after immunotherapy and disclosed that the degree of the immunotherapeutic effect is predicted by the frequencies of the CD4(+) and CD8(+) T cells producing IL-2 or TNF-α or both. Total frequencies and frequencies of double-cytokine CD4 T cell producers were enhanced by F1 and F3 vaccines. Collectively, our multifunctional analysis disclosed that immunotherapeutic protection improved as the CD4 responses progressed from 1+ to 2+, in the case of the F1 and F3 vaccines, and as the CD8 responses changed qualitatively from 1+ to 3+, mainly in the case of the F1 vaccine, providing new correlates of immunotherapeutic protection against cutaneous leishmaniasis in mice based on T-helper TH1 and CD8(+) mediated immune responses.

Dataset Information

Activity of paromomycin against Leishmania amazonensis: Direct correlation between susceptibility in vitro and the treatment outcome in vivo.

Publications

Activity of paromomycin against Leishmania amazonensis: Direct correlation between susceptibility in vitro and the treatment outcome in vivo.

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