Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Nov 10:10:595415.
doi: 10.3389/fcimb.2020.595415. eCollection 2020.

Short-Course Treatment With Imipramine Entrapped in Squalene Liposomes Results in Sterile Cure of Experimental Visceral Leishmaniasis Induced by Antimony Resistant Leishmania donovani With Increased Efficacy

Sandip Mukherjee  1 , Supratim Pradhan  2 , Souradeepa Ghosh  2 , Shyam Sundar  3 , Shantanabha Das  1 , Budhaditya Mukherjee  1   2 , Syamal Roy  1   4
Affiliations

Short-Course Treatment With Imipramine Entrapped in Squalene Liposomes Results in Sterile Cure of Experimental Visceral Leishmaniasis Induced by Antimony Resistant Leishmania donovani With Increased Efficacy

Sandip Mukherjee et al. Front Cell Infect Microbiol. .

Abstract

Previously we have shown that long term oral treatment of tricyclic-antidepressant-drug, imipramine, against experimental visceral leishmaniasis, results in clearance of organ parasites, regardless of input infection, either with antimony-sensitive (SbS) or antimony-resistant (SbR) Leishmania donovani (LD) clinical isolates. Although continuous imipramine monotherapy for 28 days (5 mg/kg) results in significant clearance of organ parasites in both SbR and SbSLD infected hamsters, the dose for the sterile parasite clearance from visceral organ is comparatively higher (10 mg/kg) and shows signs of toxicity. Hence, to reduce the toxicity, we encapsulated imipramine in squalene-phosphatidylcholine (SP) liposome (Lip-Imi) and tested its efficacy for a short-course treatment (10 days) in the animal model of visceral leishmaniasis. We observed a significant reduction of hepatic toxicity coupled with sterile parasite clearance in case of this short-course treatment of Lip-Imi, which is absent with free Imi treatment. This also correlates with significant increase in serum availability of imipramine in case of Lip-Imi treatment due to sustained release. Clearance of parasite was coupled with the polarization of antileishmanial immune repertoire from Th2 to Th1 after treatment with Lip-Imi in both SbRLD and SbSLD infected mouse models of LD infection. This study showed that imipramine is effective against both SbSLD and SbRLD at a significantly lower dose with reduced time course of treatment without any toxic side effects, when encapsulated in SP-liposome. Thus, the drug has the potential to be repurposed for the treatment of Kala-azar.

Keywords: antimony resistance; efficacy; imipramine; liposome; visceral leishmaniasis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(A, B) Measurement of size of Imipramine entrapped PC-Squalene liposomes and empty liposome by differential light scattering (DLS) method. Liposomes are prepared, diluted and scanned under optical particle analyzer. (C) Representative figure of Cryo-EM structure of Lip-Imi (panel1) and empty Imi (panel2). (D) Measurement of the amount of liposome entrapped imipramine by the quantification of supernatant imipramine. Set I, Set II and Set III represents % liposomal entrapment from 6, 8, and 10 mg imipramine, respectively. Each set represents independent quantification experiment.
Figure 2
Figure 2
Six weeks old mice/hamsters were infected with either BHU 575 (A, C) or Ag83 (B, D) LD parasites and infection was allowed to establish for next 8 weeks. Eight-week infected mice/hamsters received the following treatment: Saline (Group I), vesicles (Group II), Lip-Imi (10 mg/kg) (Group III), Lip-Imi (20 mg/kg) (Group IV), respectively at days 1, 4, 7, and 10 and group V received 10 mg/kg/day of imipramine orally for 10 days. Two days after the last treatment, mice/hamsters were sacrificed, and the hepatic and the splenic parasite load was determined by stamps-smear method and the by the limiting dilution method. Total parasite load in each organ is expressed in LDU unit. 1 LDU = amastigote per nucleated cell × organ weight in milligram. Total concentration of imipramine in the serum of mice and hamster after the completion of treatment of imipramine and liposomal imipramine (E). Results are presented as means ± SD, with statistical significance being determined with respect to infected control or between Lip-Imi and oral imipramine treatment. ***p < 0.001 and **p = 0.001–0.01 and NS-representing non-significant.
Figure 3
Figure 3
(A) Leishmania donovani (AG83 or BHU575)-infected and treated mice were sacrificed, and splenocytes were isolated and incubated with 5 µ/mL of SLA for 48 h. Levels of cytokines (i) IL-12, (ii) IFN-γ, (iii) TNF-α (iv) , and IL-10. (v) TGF-β in culture supernatants were determined by ELISA. Results are representative of one of three individual experiments. (B) Imipramine treatment favors expansion of antileishmanial T-cells. Splenocytes isolated from SbSLD (B, I) and SbRLD (B, II) LD infected (Group I), vesicles (Group II), Lip-Imi (10 mg/kg) (Group III), Lip-Imi (20 mg/kg) (Group IV), and orally 10 mg/kg for 10 day (Group V) drug treated mice were stimulated with SLA (5 µg/mL) (○) and non-specific mitogen ConA (5 µg/mL) (□), and the resulting proliferation of splenocytes was assayed using MTT cell viability assay. (C) The hepatic enzyme (ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase) and serum creatinine level in untreated uninfected, untreated infected, and imipramine treated infected hamsters.For A, results are presented as means ± SD, with statistical significance being determined with respect to infected control or between Lip-Imi and oral imipramine treatment. ***p < 0.001. For (B), experiments were repeated thrice one representative data is shown. For C, results are presented as means ± SD, with statistical significance being determined with respect to normal uninfected or LD infected animals in presence or absence of Lip-Imi treatment. *p = 0.01–0.05.
Figure 4
Figure 4
(A) Long term survival of normal, BHU 575 infected, oral 10 days imipramine treated and Lip-Imi (10 mg/kg) treated infected hamsters in terms of % survival. 20 hamsters from each group were used for the study. (B) Photographic representation (×40), of enhanced granuloma development (black arrow, right panel) in Lip-Imi (10 mg/kg) treated hamsters as opposed to immature granuloma (black arrow, left panel) formation in Imi (10 mg/kg) treated hamsters following infection with BHU575.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Alving C. R., Steck E. A., Chapman W. L., Waits V. B., Hendricks L. D., Swartz G. M., et al. (1978). Therapy of leishmaniasis: superior efficacies of liposome-encapsulated drugs. Proc. Natl. Acad. Sci. 75 (6), 2959–2963. 10.1073/pnas.75.6.2959 - DOI - PMC - PubMed
    1. Andrade-Neto V. V., Pereira T. M., do Canto-Cavalheiro M., Torres-Santos E. C. (2016). Imipramine alters the sterol profile in Leishmania amazonensis and increases its sensitivity to miconazole. Parasites Vectors 9 (1), 183. 10.1186/s13071-016-1467-8 - DOI - PMC - PubMed
    1. Badiee A., Jaafari M. R., Khamesipour A. (2007). Leishmania major: immune response in BALB/c mice immunized with stress-inducible protein 1 encapsulated in liposomes. Exp. Parasitol 115 (2), 127–134. 10.1016/j.exppara.2006.07.002 - DOI - PubMed
    1. Barratt G., Bretagne S. (2007). Optimizing efficacy of Amphotericin B through nanomodification. Int. J. Nanomed 2 (3), 301–313. - PMC - PubMed
    1. Basu R., Bhaumik S., Basu J. M., Naskar K., De T., Roy S. (2005). Kinetoplastid membrane protein-11 DNA vaccination induces complete protection against both pentavalent antimonial-sensitive and -resistant strains of Leishmania donovani that correlates with inducible nitric oxide synthase activity and IL-4 generation: evidence for mixed Th1- and Th2-like responses in visceral leishmaniasis. J. Immunol. 174 (11), 7160–7171. 10.4049/jimmunol.174.11.7160 - DOI - PubMed

Publication types