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Table of Contents
RESEARCH ARTICLE
Year : 2020  |  Volume : 57  |  Issue : 1  |  Page : 78-84

Study of the in vitro and in vivo antileishmanial activities of nimodipine in susceptible BALB/c mice


1 Research Center for Skin Diseases and Cutaneous Leishmaniasis; Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
2 School of Pharmacy; Mashhad University of Medical Sciences, Mashhad, Iran
3 School of Pharmacy; Nanotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
4 Research Center for Skin Diseases and Cutaneous Leishmaniasis, Mashhad, Iran
5 School of Pharmacy; Nanotechnology Research Center; Department of Pharmaceutical Nanotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

Date of Submission27-Aug-2018
Date of Acceptance15-Nov-2018
Date of Web Publication05-Feb-2021

Correspondence Address:
Dr Mahmoud Reza Jaafari
School of Pharmacy, Mashhad University of Medical Sciences, Mashhad
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.308805

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  Abstract 

Background & objectives: Pentavalent antimonials are the standard treatment for cutaneous leishmaniasis (CL), however, treatment failures are frequent. Nimodipine, a calcium channel blocker is known to show promising antiprotozoal effects. Here, we investigated the antileishmanial effect of Nimodipine in both in vitro and in vivo BALB/c mice model of CL. We also compared the in vivo effect with amphotericin B and meglumine antimoniate in the experimental CL mice model.
Methods: Colorimetric alamar blue assay and J774 A.1 mouse macrophage cells were used to determine the effect of nimodipine on promastigotes and amastigotes viability, respectively. Then, the in vivo activity of nimodipine was compared to that of conventional therapies in both the early and established courses of Leishmania major infection in susceptible non-healing BALB/c mice.
Results: Nimodipine was highly active against promastigotes and amastigotes of L. major with IC50 values of 49.40 and 15.03 μM, respectively. In the early model, the combination therapy with meglumine antimoniate and nimodipine showed no parasites in the spleen or footpad of animals. The footpad thickness was significantly lower in mice treated with either nimodipine (1 mg/kg or 2.5 mg/kg) or amphotericin B compared to the control group in the established lesions model. However, no complete remission was observed in the footpad lesion of any of the treatment groups (nimodipine, amphotericin B, meglumine antimoniate, and combination therapy).
Interpretation & conclusion: The effect of nimodipine was comparable to that of amphotericin B and meglumine antimoniate in early and established CL lesion models. Since nimodipine is more cost-effective than conventional therapies, our results merit further investigation in other animal models and voluntary human subjects.

Keywords: Amastigote; BALB/c mice; Leishmania major; meglumine antimoniate; nimodipine; promastigote


How to cite this article:
Azadi R, Alipour-Talesh G, Yazdanpanah MJ, Alavizadeh SH, Maleki M, Banihashemi M, Jaafari MR. Study of the in vitro and in vivo antileishmanial activities of nimodipine in susceptible BALB/c mice. J Vector Borne Dis 2020;57:78-84

How to cite this URL:
Azadi R, Alipour-Talesh G, Yazdanpanah MJ, Alavizadeh SH, Maleki M, Banihashemi M, Jaafari MR. Study of the in vitro and in vivo antileishmanial activities of nimodipine in susceptible BALB/c mice. J Vector Borne Dis [serial online] 2020 [cited 2021 Apr 18];57:78-84. Available from: https://www.jvbd.org/text.asp?2020/57/1/78/308805


  Introduction Top


Leishmaniasis has diverse clinical manifestations and is caused by different species of the genus Leishmania. Countries with the highest estimated case counts of cutaneous leishmaniasis (CL) include Afghanistan, Algeria, Brazil, Colombia, the Islamic Republic of Iran, Pakistan, Peru, Saudi Arabia, and the Syrian Arab Republic (WHO Report 2018). The annual incidence of leishmaniasis is about 2 million cases worldwide (1.5 million CL; 0.5 million visceral leishmaniasis)[1],[2]. The relatively high prevalence of CL infection in several parts of Iran has made its control a priority for public health organizations[3],[4]. Despite the serious side effects such as renal and cardiac toxicity, antimonials remained the standard treatment for leishmaniasis in endemic countries. However, treatment failure is common in the endemic areas and resistance has been frequently reported[4],[5],[6],[7]. Thus, there is an urgent need for new mono- or combination therapies of traditional treatment in case of resistant cases.

Calcium channel blockers are a class of drugs originally developed to treat hypertension and vascular spasms[8]. It is known that some calcium channel blockers such as verapamil can increase the susceptibility of Leishmania promastigotes and amastigotes to antimonials[9],[10],[11]. Nimodipine, a 1,4-dihydropyridine calcium channel blocker, is currently used to prevent ischemic damage caused by vasospasm in patients with subarachnoid haemorrhage[8]. Tempone et al[12] have demonstrated that nimodipine has promising leishmanicidal activity against promastigotes and amastigotes of Leishmania chagasi (the protozoan causing visceral leishmaniasis in Latin America)[13],[14]. In the search for a novel drug to treat resistant cases of CL, we aimed at studying the antileishmanial activities of nimodipine against promastigotes and amastigotes of L. major (a causative agent of zoonotic CL in Iran) in culture media. We also studied the in vivo efficacy of nimodipine alone or in combination with meglumine antimoniate in both early and established cutaneous Leishmania lesion-BALB/c mice model of leishmaniasis.


  Material & Methods Top


Drugs

Nimodipine vials (0.02% solutions (10 mg/50 ml)) manufactured by M/s. Bayer, Germany as Nimotop®, meglumine antimoniate (1500 mg/5ml) manufactured by M/s. Strop, Belgium as Meglusan®, amphotericin B vials (50 mg) manufactured by M/s. Cipla, India and dextrose 5% in water (D5W) manufactured by M/s. Samen Pharmaceutical Co. Iran were used in this study.

Animals

Female BALB/c mice, 4–6 wk-old, were purchased from the Pasteur Institute (Tehran, Iran). Animals were kept in the animal house of Pharmaceutical Research Centre with 12/12 h light/dark cycle at 21°C. They had free access to tap water and standard laboratory pellet chow (Khorassan Javane Co., Mashhad, Iran).

Parasite and macrophages

The virulence of L. major strain MRHO/IR/75/ER was maintained by a passage in BALB/c mice. The amastigotes were isolated from the spleen of an infected mouse and then cultured on Novy-MacNeal-Nicolle (NNN) medium and subcultured in RPMI 1640 (Sigma) containing 10% (v/v) heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, and 100μg/ ml streptomycin sulfate (RPMI-FCS) at 25 ± 1°C. The J774 A.1 mouse macrophage cell lines were purchased from the Pasteur Institute of Iran (Tehran, Iran) and maintained in DMEM containing 10% FBS at 37°C in a 5% CO2 atmosphere.

In vitro cytotoxicity assay using promastigotes

Alamar blue assay was used to determine the effect of nimodipine on the viability of Leishmania promastigotes. The effect of the drug on the viability of Leishmania promastigotes was determined by monitoring the absorbance of alamarBlue at two wavelengths after a 48 h culture period in the presence of different concentrations of the drug. Parasites harvested at stationary phase (2.5×106 promastigotes) were added to each well of 96-well flat-bottom plates containing different concentrations of nimodipine in triplicate. AlamarBlue (20 μl/well) was added to each well and the plate was then incubated for 48 h at 25°C. Blank wells consisted of 100 μl RPMI+FCS and 100 μl nimodipine at 100 μg/ml with no cell.

Positive control wells, indicating survival of parasites without any drug, consisted of 100 μl RPMI+FCS and 100 μl of the promastigotes. The relative absorbance was correlated to the quantity of promastigotes per well by calculating the percent of reduction when the samples were read at 570 and 600 nm. The half-maximal inhibitory concentration (IC50) for nimodipine was calculated by CalcuSyn software version 2.1 (Biosoft, Cambridge, UK)[15].

In vitro cytotoxicity assay using amastigotes

The J774 A.1 mouse macrophage cells were dispensed at a concentration of 50,000 macrophages per well into eight-well Lab-Tek (Nunc) chamber slides and kept at 37°C in 5% CO2 for 24 h. Cells were then infected with L. major promastigotes at a ratio of 7 promastigotes per macrophage and incubated at 37°C in 5% CO2 for 24 h to allow parasite internalization into the cells. Parasite excess was removed by gently washing the cells with PBS three times, and the infected cells were incubated for 24 h for a second time to allow the establishment of the infection. This was followed by exposing the cells to different concentrations of nimodipine in triplicate for 2 days. The experiment was ended by methanol fixation of the slides. Afterward, the slides were stained with Giemsa and evaluated microscopically to calculate the percentage of infected cells. The IC50 for nimodipine was calculated by CalcuSyn software version 2.1 (Biosoft, Cambridge, UK)[15],[16],[17].

Effect of nimodipine on early L. major lesion model in susceptible BALB/c mice

The left footpad of 49 female BALB/c mice, 7 per group, was subcutaneously inoculated on Day 0 with L. major promastigotes (2 × 106 in 50 ml) harvested at stationary phase[17]. Mice were randomly divided into 7 groups receiving one of the following intraperitoneal (ip) regimens on Days 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 post-parasite inoculation: nimodipine (1 mg/kg), nimodipine (2.5 mg/kg), nimodipine (5 mg/kg), meglumine antimoniate (100 mg/kg), amphotericin B (1 mg/kg), nimodipine (2.5 mg/ kg) + meglumine antimoniate (100 mg/kg) (injected separately) and dextrose 5% (0.2 ml). Treatment outcome in each group was determined by monitoring footpad inflammation and a load of parasites in the spleen and footpad of infected mice as described later.

Effect of nimodipine in established L. major lesion model in susceptible BALB/c mice

Female BALB/c mice, 6 per group, were subcutaneously inoculated in the left footpad with L. major promastigotes (2 × 106 in 50 ml) harvested at stationary phase[17]. The randomly divided 6 groups of mice received one of the following ip regimens on Days 27, 29, 31, 33, 35, 37, 39, 41, 43 and 45 post-parasite inoculation: nimodipine (1 mg/kg), nimodipine (2.5 mg/kg), nimodipine (5 mg/kg), meglumine antimoniate (100 mg/kg), amphotericin B (1 mg/kg), and nimodipine (2.5 mg/kg)+meglumine antimoniate (100 mg/kg).

Footpad swelling

Lesion development in different groups was monitored by measuring footpad swelling (thickness) using a metric caliper (Mitutoyo, Japan)[17] three times weekly. Lesion size was determined by subtracting the thickness of uninfected contralateral footpad from the infected one. Each mouse was marked by a picric acid solution to be differentiated from others in the same group.

Quantitative parasite burden

The number of viable L. major parasites in the spleen and footpad of each group was determined using limiting dilution assay[17],[18]. Three mice in each group were sacrificed by cervical dislocation on Days 21 (early CL lesion model) and 48 (established CL lesion model) after promastigotes inoculation and the spleens and footpads were aseptically isolated. The spleens were homogenized in 1 ml RPMI-FCS with a sterile syringe piston.

The infected footpads were transferred into the tubes containing 1 ml RPMI-FCS, and a certain amount of zirconium beads. The samples were completely homogenized using a bead beater for 20 sec in one cycle. The homogenates were diluted with the same media in 8 serial 10-fold dilutions in each well of 96-well flat-bottom microtitre plates containing a solid layer of rabbit blood agar in triplicate and kept at 25 ± 1°C for 7 days.

The positive (containing motile parasite) and negative (without motile parasite) wells were examined using an inverted microscope (CETI, UK). Parasite burden in the spleen and foot of each mouse was calculated[14],[18]. The data are reported as the mean and standard error of the mean of the last positive well-multiplied by the dilution factor[19],[20],[21].

Statistical analysis

GraphPad Prism, version 5.0 for Windows, (San Diego, California, USA) was used for statistical analysis and graph preparation. A one-way ANOVA statistical test was used to assess the significance of the differences among different groups. Results with p <0.05 were considered to be statistically significant.

Ethical statement

The animal experiments were carried out according to the Ethical Committee Acts of Mashhad University of Medical Sciences, Mashhad, Iran (Grant No. 900050; October 27, 2011).


  Results Top


Effects of nimodipine on L. major promastigotes in vitro

The IC50 of nimodipine against L. major promastigotes was 49.40 μM (at 95% CI: 40.72–59.94 μM).

Intracellular drug sensitivity assay

To examine the efficacy of nimodipine on intracellular L. major amastigotes, the macrophage cell line J774.1A was infected with L. major at a ratio of 7:1, parasite to macrophage. Nimodipine was active against intracellular amastigotes of L. major with an IC50 value of 15.03 μM (95% CI: 4.23–53.39 μM). We observed macrophage necrosis in cells treated with nimodipine at concentrations higher than 50 μg/ml which indicate possible toxicity of the drug on mammalian cells at those concentrations.

Antileishmanial activity in the early CL lesion model in BALB/c mice

In this lesion model, different formulations were administered ip on Days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 post-parasite inoculations. No statistically significant difference was observed in the size of lesion of animals treated with nimodipine 2.5 mg/kg, nimodipine 5 mg/kg, meglumine antimoniate 100 mg/kg, amphotericin B 1 mg/kg, nimodipine 2.5 mg/kg + meglumine antimoniate 100 mg/ kg or Control group (p >0.05) throughout the study period. Nimodipine at a dose of 2.5 mg/kg or amphotericin B (1 mg/kg) significantly decreased footpad thickness compared to nimodipine at a dose of 1 mg/kg on Days 20 and 22 (p <0.05).

Administration of nimodipine at a dose of 5 mg/kg caused significantly smaller footpad swelling compared to nimodipine 1 mg/kg on Days 17 to 31 (p <0.05). Footpad swelling was also significantly smaller when meglumine antimoniate was used alone or in combination with nimodipine 2.5 mg/kg as compared to nimodipine 1 mg/kg (p <0.05) [Figure 1].
Figure 1: In vivo antileishmanial activity in early cutaneous L. major-infected BALB/c mice. Different formulations were administered ip on Days 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 post-parasite inoculation. Values are the means ± standard errors of the means (n = 7). On Days 20 and 22 mice treated with nimodipine 2.5 mg/kg, nimodipine 5 mg/kg, amphotericin B 1 mg/kg and nimodipine 2.5 mg/kg + meglumine antimoniate 100 mg/kg, had a significantly lower* (p <0.05) footpad thickness compared to nimodipine at 1 mg/kg.

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Splenic and footpad parasite burden in early CL lesion model

At week 3 of post-parasite inoculation, the spleen of animals in nimodipine (1 and 2.5 mg/kg), amphotericin B (1 mg/kg), nimodipine 2.5 mg/kg + meglumine antimoniate (100 mg/kg) (p <0.01) and meglumine antimoniate (100 mg/kg) (p < 0.05) groups showed significantly lower number of parasites compared to the control group [Figure 2]a. At week 3 post-parasite inoculation, the footpad parasite burden in the mice treated with nimodipine 2.5 mg/kg + meglumine antimoniate (100 mg/kg) was significantly lower than that of all other groups (p <0.001). Significant differences were not observed among other groups [Figure 2]b.
Figure 2: Splenic and footpad parasite burden in early cutaneous L. major BALB/c mice. Formulations were administered ip on Days 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 after parasite injection. The number of viable L. major in the: (a) spleens; and (b) footpad of groups of mice was quantified by limiting dilution assay on Day 21 after inoculation of L. major promastigotes. Values are means ± standard errors of the means (n = 3). **(p <0.01), *(p <0.05) groups compared to the (a) control; ***(p <0.001) indicates nimodipine 2.5 mg/kg + meglumine antimoniate 100 mg/kg compared to (b) all other groups.

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Antileishmanial activity in established CL lesion model in BALB/c mice

In this model of established lesion, treatment was initiated after the emergence of lesions (27 days after parasite injection). Drugs were administered ip on Days 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45 after parasite injection. As demonstrated in [Figure 3], there were no significant differences (p >0.05) in the footpad swelling among different groups before the initiation of therapy (Day 27 post-parasite inoculation). Following the start of treatment, footpad swelling decreased in some of the treatment groups. Mice treated with nimodipine at a dose of 1 mg/kg, demonstrated a significantly smaller footpad swelling versus the control group (p <0.05). From Day 41 onward, significantly smaller footpad thickness was observed in the groups of mice administered with nimodipine 1 mg/kg, nimodipine 2.5 mg/kg, and amphotericin B 1 mg/kg as compared to the control group (p <0.05). This was while there was no significant difference in the footpad swelling of animals treated with meglumine antimoniate alone or in combination with nimodipine 2.5 mg/kg as compared to the control group (p >0.05).

Splenic and footpad parasite burden in established CL lesion model

No significant difference was observed among different treatment groups in this treatment strategy (p >0.05). On Day 48 of post-parasite inoculation, no significant difference was detected among the groups treated with nimodipine 2.5 mg/kg, meglumine antimoniate 100 mg/kg, amphotericin B 1 mg/kg, nimodipine 2.5 mg/ kg + meglumine antimoniate 100 mg/kg or the control group, however, the footpad parasite number of mice treated with nimodipine 1 mg/kg was significantly reduced compared to all other groups (p < 0.01).


  Discussion Top


The present study assessed the in vitro and in vivo effects of nimodipine against L. major infection. We observed that nimodipine was highly active against promastigotes and amastigotes of L. major. Early stage treatment with nimodipine, meglumine antimoniate, or amphotericin B, did not have any significant effect on the footpad swelling. Nonetheless, early combination therapy using meglumine antimoniate and nimodipine induced no parasite in mice spleens or footpad. In the setting of established lesion, both doses of nimodipine (1 and 2.5 mg/ kg) and also amphotericin B reduced the footpad thickness but did not induce complete remission.

Indeed, calcium channel blockers are known for their effects on arterial smooth muscles. They are widely used in the treatment of hypertension or conditions associated with arterial spasm such as subarachnoid haemorrhage[8]. There are reports indicating that these compounds may have certain antimicrobial and antiparasitic activities. Misra et al[22] have demonstrated the inhibitory effects of Nifedipine and verapamil on L. donovani-macrophage attachment, suggesting the role of Ca2+ ion in the invasion process of the organism. There are also several reports that verapamil can increase the in vitro susceptibility of promastigotes and amastigotes of L. donovani[9],[11], and L. tropica[10] to antimonial drugs which can reverse their resistance to therapy.

The 1, 4-dihydropyridine calcium channel blockers have presented activity against cutaneous and visceral species of Leishmania. Palit and Ali reported that lacidipine, amlodipine, verapamil, and diltiazem could all inhibit Ca2+ uptake by Leishmania, but only lacidipine and amlodipine showed in vivo antivisceral leishmaniasis activity[23].

Tempone et al[12] have demonstrated that nimodipine was effective against promastigotes and intracellular amastigotes of L. chagasi, with IC50 of 81.2 and 21.5 μM, respectively. Nimodipine showed approximately 4-fold higher potency against amastigotes compared to the standard treatment with pentavalent antimony. Nimodipine at a dose of 31 to 128 μM was effective against promastigotes of L. amazonensis and L. major as well. The in vitro activity against promastigotes showed that L. major was the most susceptible subgenus to nimodipine, with IC50 of 31 μM[12].

In the present study, we observed that nimodipine was active against promastigotes of L. major with an IC50 value of 20.67 μg/ml (49.40 μM). Intracellular amastigotes of L. major were even more sensitive (about three times) to nimodipine with the IC50 value of 6.29 μg/ml (15.03 μM). In a previous study, we have shown that pentavalent antimonials have no effect on promastigote growth while ED50 of liposomal meglumine antimoniate against intracellular amastigotes was 28.9 μg/ml[24]. We have also shown that the ED50 of Fungizone™, the traditional formulation of AmB as a mixed micelle with deoxycholate, against the promastigote and intracellular amastigote forms of the parasites were 0.24 and 0.108 μg/ml, respectively. The ED50 of Fungizone™ against promastigotes was greater when tested against the amastigotes[25]. Similarly, others reported that intramacrophage amastigotes are more susceptible to different liposomal formulations of amphotericin B than promastigotes[26].

Previous pre-clinical studies have mostly focused on promastigotes and less commonly on amastigotes of Leishmania species[9],[10] and in vivo drug susceptibility studies are rare. Our encouraging in vitro results prompted us to evaluate the in vivo effect of different doses of nimodipine in L. major-BALB/c mouse model of leishmaniasis and to compare it with amphotericin B and meglumine antimoniate.

The results of the in vivo study in the early CL lesion model showed that the combination of nimodipine with meglumine antimoniate significantly reduced parasite load in the spleen or footpad of mice and this effect was more than either of individual treatments in the early CL lesion model. These findings indicated some degree of synergistic effects between nimodipine and meglumine antimoniate on footpad parasite burden in the early stages of CL. While there was no significant difference in the footpad thickness of meglumine antimoniate and the control group in the established lesion model, the footpad thickness of mice treated with either dose of nimodipine (1 mg/kg or 2.5 mg/kg) and amphotericin B was significantly lower than that of the control mice with established lesions.

Evaluating the in vivo effects of nimodipine in both early and established lesion models in susceptible BALB/c mice showed that the efficiency does not correlate with the dose. Considering all the results, it seems that the effect of nimodipine is comparable to that of amphotericin B and meglumine antimoniate in both lesion models of leishmaniasis. The results of lesion size showed no complete cure in any of the treatment groups (nimodipine, amphotericin B, meglumine antimoniate, or combination therapy). This is most probably due to the fact that BALB/c mice are the most susceptible strains to L. major infection and conventional treatments are not fully effective in this strain of mice.

In most cases, L. major infection in BALB/c mice results in progressive lesions with increased parasite replications, which is partly dependent on CD4+ TH2 type response[27]. However, nimodipine may be useful in the treatment of CL in humans, as humans, unlike BALB/c mice, are resistant to leishmaniasis.


  Conclusion Top


To our knowledge, this study provides the first report on both in vitro and in vivo effects of nimodipine in early and established CL lesion models. Early-stage combination therapy using meglumine antimoniate and nimodipine induced no parasites in mice spleens or footpads. On the other hand, in the established course of L. major infection, treatment with both 1 and 2.5 mg/kg doses of nimodipine and also amphotericin B reduced footpad thickness compared to the control group.

However, no complete remission was observed in the footpad lesion of any of the treatment groups (nimodipine, amphotericin B, meglumine antimoniate, and combination therapy). Overall, the effect of nimodipine was comparable to that of amphotericin B and meglumine antimoniate in early and established CL lesion models.

In conclusion, nimodipine is active against promastigotes and amastigotes of L. major in vitro and in vivo effect in the course of leishmaniasis in BALB/c mouse model is not the same in early and late stages of infection. Since the production of nimodipine production is less costly than conventional therapies, we believe our results merit further investigation in other animal models and voluntary human subjects with CL.


  Acknowledgements Top


This project was supported by a grant from the Vice-Chancellor for Research, Mashhad University of Medical Sciences, Mashhad, Iran.

Conflict of interest

The authors declare that they have no conflict of interest in this study. The funder had no role in the study design, data collection, analysis, the decision to publication or preparation of the manuscripts.

 
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