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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 9  |  Issue : 1  |  Page : 31-36

Antischistosomal activity of Mirazid in experimental schistosomiasis mansoni: exploring the controversy


Department of Parasitology, Medical Research Institute, Alexandria University, Alexandria, Egypt

Date of Submission14-Dec-2015
Date of Acceptance22-Mar-2016
Date of Web Publication25-Oct-2016

Correspondence Address:
Mohammad A Al-Kazzaz
27 Street, 15 Al-Awayed, Alexandria
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1687-7942.192993

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  Abstract 

Background
Mirazid (MZD) was licensed in Egypt for treatment of schistosomiasis in the year 2002; the drug gained much attention experimentally and clinically, with conflicting views on its efficacy.
Objective
The study aimed to evaluate MZD anti-schistosomal activity in an animal model at a selected dose.
Materials and methods
Swiss albino mice (n=36) were infected with Schistosoma mansoni and divided into two equal groups of 18 mice each: group 1 was the nontreated infected control group given only the vehicle; gourp 2 was infected and treated with MZD at a dose of 500 mg/kg for 5 days per os 7 weeks postinfection. Efficacy of the drug was assessed parasitologically with fecal egg counts evaluated every other day until the animal was euthanized at 1, 2, and 4 weeks post-treatment (WPT); worm burden, tissue egg count and oogram pattern were studied at 1, 2, and 4 WPT.
Results
MZD reduced fecal egg counts in infected mice (69.6%) and reduced total worm load (71.9%) and tissue egg counts in the intestine and the liver (66 and 77.4%, respectively) at 4 WPT. The drug changed oogram pattern with progress of treatment.
Conclusion
MZD has moderate antischistosomal activity in animal models.

Keywords: mice, mirazid, oogram pattern, Schistosoma mansoni, tissue egg count, worm burden


How to cite this article:
Al-Kazzaz MA, El-Sayad MH, Abu-Helw SA. Antischistosomal activity of Mirazid in experimental schistosomiasis mansoni: exploring the controversy. Parasitol United J 2016;9:31-6

How to cite this URL:
Al-Kazzaz MA, El-Sayad MH, Abu-Helw SA. Antischistosomal activity of Mirazid in experimental schistosomiasis mansoni: exploring the controversy. Parasitol United J [serial online] 2016 [cited 2023 Nov 29];9:31-6. Available from: http://www.new.puj.eg.net/text.asp?2016/9/1/31/192993


  Introduction Top


Mirazid (MZD) is a natural pharmaceutical preparation introduced into the Egyptian market by Pharco Pharmaceuticals (Alexandria, Egypt) in the form of soft gelatin capsules since 2002. Each capsule contains 300 mg of the purified oleoresin extract of Commiphora molmol (Myrrh) [1]. The Egyptian drug authority in the Egyptian Ministry of Health registered this product for treatment of schistosomiasis (Reg. No. 21655/2002) [2]. The antischistosomal properties of MZD were investigated, and reviewed both experimentally and clinically with controversy regarding its efficacy [3].

In humans, the efficacy of MZD as an anti-schistosomal drug was evaluated clinically with positive results [4],[5],[6],[7],[8],[9]. All in all, they enrolled 365 schistosomiasis patients (adults and children) and treated them with MZD at a dose of 10–11.5 mg/kg for 3–6 days. The cure rate, evaluated parasitologically (disappearance of fecal eggs) by Kato–Katz and rectal snip techniques at 2–3 months after treatment, was 80.7–100%. The drug proved to have nonsignificant side effects. The drug was tested by other researchers, with disappointing results. It showed little or no beneficial activity as reported by Barakat et al. [10], Botros et al. [11], and Osman et al. [12], who altogether enrolled 206 patients (adults or children) treated with MZD at a dose of 300–600 mg daily for 3–6 days. The drug showed a cure rate that ranged from 3.7 to 15.6 % at 3–8 WPT.

Such controversy prompted us to study the efficacy of MZD experimentally as there are many parasitological parameters other than fecal egg counts.


  Materials and methods Top


Experimental study

The study was conducted at the Department of Parasitology, Medical Research Institute (MRI), Alexandria University, Alexandria, Egypt, during the period from 21 July 2013 to 14 October 2014.

Materials

The study included 36, 8-week-old female Swiss albino mice of the CD-1 strain weighing 20±2 g, obtained from the animal house of the MRI, Alexandria University, Alexandria, Egypt. Laboratory-bred Biomphalaria alexandrina snails infected with miracidiae of the Egyptian (CD) strain of Schistosoma mansoni were obtained from the Schistosome Biologic Supply Center, Theodore Bilharz Research Institute, Cairo, Egypt. MZD capsules were obtained as free medical samples from Pharco Pharmaceuticals, Batch No: 296. Each capsule (300 mg) was emptied in a flask containing 3 ml of 4% Cremophor EL used as the vehicle for drug administration.

Methods

Cercarial shedding

Infected B. alexandrina snails were washed with dechlorinated water and kept in an aerated aquarium (using an electric pump) in a dark place (by covering the glass bath with a black plastic bag) [13]. Before use, snails were rinsed gently with a small volume of water to remove feces and other debris, then resuspended in water (1 ml/snail) and left uncovered in a glass test tube under white fluorescent light for a period of 30–60 min to release cercariae ([Figure 1];1). After shaking gently to ensure homogenous distribution of cercariae, 1 ml of cercarial suspension was pipetted and placed on glass slides; a drop of iodine was added to each slide to kill and stain the cercariae ([Figure 1];2). With the aid of a stereobinocular microscope, the number of cercariae was counted in each slide. Generally, three counts were made in 3 ml cercarial suspension and the average number per 1 ml was calculated.
Figure 1 Steps of mice infections: (1) cercarial shedding; (2) cercarial count; (3) cercarial inoculation.

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Experimental infection

Mice were infected using the paddling technique according to Smithers and Terry [14]. Each mouse was exposed separately to about 100 S. mansoni cercariae ([Figure 1];3). Infected mice were then segregated in separate stainless steel wire mesh cages, and received a standard well-balanced diet and water. The mice were housed in a room under controlled environmental temperature. Stool examination was performed 50 days after cercarial infection to determine the presence of S. mansoni eggs.

Drug administration

MZD was administered at a dose of 500 mg/kg for 5 days per os, 7 WPI. Each mouse required 0.1 ml solution to reach a dose of 500 mg/kg. The dose was selected as specified by Botros et al. [1] and Massoud et al. [15] and is four-fold the therapeutic dose in mice (125 mg/kg) based on Food and Drug Administration guidelines by converting the human dose to those for experimental animals.

Evaluation of drug efficacy was based on the following parasitological parameters: (a) fecal eggs were counted every other day starting 2 days post-treatment (PT) and continued until the mice were sacrificed at 1, 2, and 4 weeks post-treatment (WPT) according to Sewify [16]; (b) for recovery of adult worms, the perfusion technique was carried out according to Smithers and Terry [14]. The collected worms were counted to estimate the total worm count and differentiation into male and female worms; (c) tissue egg counts in the liver and intestine were calculated according to Cheever [17]. Tissues were frozen until examination; (d) the oogram pattern (percentage of egg developmental stages) was studied according to the method of Pellegrino et al. [18].

Statistical analysis

The data were coded, collected, tabulated, and analyzed using the independent two-sample t-test with Minitab statistical software, version 14 (Minitab Inc., Pennsylvania State College, Pennsylvania, USA). Descriptive statistics were expressed as arithmetic mean ± SD as measures of central tendency and dispersion, respectively. The level of significance (P<0.05) was considered statistically significant. The change% in each parasitological parameter was calculated according to the following equation.



Ethical considerations

The study protocol was reviewed and approved by the Ethics Committee of the MRI, University of Alexandria.


  Results Top


MZD resulted in a nonsignificant reduction in the number of S. mansoni eggs in the stool of the treated group (4.5%) 1 WPT followed by significant reduction of 39.5 and 69.6% at 2 and 4 WPT, respectively, compared with the nontreated infected group ([Figure 2] and [Table 1]). MZD significantly reduced the total worm burden by 34, 50, and 72% at 1, 2, and 4 WPT, respectively ([Table 1]). The number of male and female worms decreased, males being more affected than females at 1 and 2 WPT with significant reduction of 37.9 and 56.7% compared with 25.3 and 36.8%, respectively. At 4 WPT, there was significant reduction of both male (75.1%) and female worms (60%) ([Table 1]).
Figure 2 Egg counts in the stool of MZD-treated Schistosoma mansoni-infected mice compared with nontreated infected mice. MZD, mirazid.

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Table 1 Effect of mirazid on fecal egg counts, total worm burden, and worm sex in Schistosoma mansoni-infected treated mice compared with nontreated infected mice according to WPT

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MZD produced significant reduction in the tissue egg counts in the intestine (29.1–66%) throughout the follow-up weeks. Reduction in the hepatic tissue egg load was nonsignificant at 1 WPT (22.1%), and became significant in the second and fourth week (42.7 and 77.4%) ([Table 2]). In the treated group the percentage of dead eggs at 1, 2, and 4 WPT and the mean percentage of mature eggs at 2 and 4 WPT were significantly increased compared with the values in nontreated infected mice. At 4 WPT, more than 50% of the eggs were mature, which denotes interruption in egg laying as the number of immature eggs decreased with increase in the number of mature and dead eggs.
Table 2 Effect of mirazid on the mean tissue egg counts and the oogram pattern in Schistosoma mansoni-infected mice at 1, 2, and 4 WPT compared with nontreated mice

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  Discussion Top


MZD was tested in in vitro studies by Hassan et al. [19], Sharaf [20], and Bakr et al. [21] in which they exposed S. mansoni worms to various concentrations of MZD from 100 to 400 µg/ml. The drug elicited maximal somatic muscle contraction at the highest concentration and a strong lethal effect occurred after 24 h exposure. Karamustafa et al. [22] verified the lethal effects of MZD on adult worms and larvae of S. mansoni (IC50=7.18–32.69 μg/ml).

In our in vivo study, MZD was tested experimentally in S. mansoni-infected mice at a selected dose of 500 mg/kg for 5 days, producing a significant gradual decrease in the total number of worms up to 72% by the fourth WPT but did not abolish the infection. Using the same dose for 5 days, Massoud et al. [15] had reported a 98% reduction in total worm load 45 days post-infection, whereas Botros et al. [1] found that the drug showed only 9% total worm reduction. Bakr et al. [21] also used the same dose of 500 mg/kg but for 3 days and obtained 82.5% reduction in the total worm load at 1 month PT. Hamed and Hetta [23] reported that a dose of 600 mg/kg for 3 days led to 81.1% worm reduction 27 days PT. Shorter periods of effective response to MZD treatment were reported by Badria et al. [24] who used the drug at a dose of 500 mg/kg twice a day for 3 days and recorded 75% worm burden reduction after 5 days PT. Lotfy et al. [25] also used a dose of 600 mg/kg for 5 days with significant worm load reduction (69.3%) at 4 WPT. El-Gamal et al. [26] studied the effect of a single dose administered 6 weeks after infection and the drug eradicated 58.6% of total worms at 2 WPT. However, contradictory results reported [1],[27],[28],[29],[30],[31],[32],[33] showed low worm burden reduction rates, not more than 10%, in S. mansoni, S. haematobium, and S. japonicum-infected mice or hamsters treated with oral doses of MZD that varied from 250 to 500 mg/kg for 2–5 days. Such controversial results are expected because of variations in drug dosage and/or number of days of administration.

In the present work, the number of male and female worms decreased, males being more affected than females at 1 and 2 WPT with significant reduction of 37.9 and 56.7% compared with 25.3 and 36.8%, respectively. At 4 WPT, there was significant reduction in the number of both male and female worms (66%). These results apparently conform with those of Bakr et al. [21] for male worms (75.1%) but not for female worms (83.8%) from MZD-treated mice 4 WPT with 500 mg/kg for 3 days; but Botros et al. [1] found nonsignificant reduction in male and female worms at 2 WPT (12.4 and 24.5%), respectively.

Relative to the decrease in the number of worms the drug resulted in nonsignificant reduction in the number of S. mansoni eggs in the stool (4.5%) at 1 WPT, but it caused significant reduction (39.6 and 69.6%) at 2 and 4 WPT compared with the non-treated infected group. Another experimental report [34] recorded 6.6% followed by 44% reduction in fecal egg counts at 2 and 4 WPT, respectively, using 500 mg/kg MZD for 5 days. In contrast, using a higher dose of 600 mg/kg for 6 days, 97% reduction in fecal egg counts was induced after 4 weeks of MZD therapy [25].

MZD resulted in significant reduction in tissue egg counts in the intestines (48.9–66%) throughout the 4 WPT follow-up in our study. Nonsignificant reduction in the hepatic tissue egg load was observed at 1 WPT (22.1%) but the drug was able to significantly reduce the egg count in the second and fourth WPT (42.7 and 77.4%, respectively). Higher tissue egg count reduction of 98.2 and 97% in the intestinal and hepatic tissue egg counts, respectively, was recorded at 5 WPT [15]. In contrast, another research [21] recorded a lower percentage reduction of 41% and 28.9% in hepatic and intestinal tissue egg counts at 4 WPT and with a similar MZD dose of 500 mg/kg, but for 3 days. Other studies [1],[27] found nonsignificant change in both intestinal and hepatic tissue egg loads at 2 WPT with a dose of MZD 300 or 500 mg/kg for 3 or 5 days, and examined 7 WPI.Regarding the oogram pattern, MZD resulted in significant increase in the percentage of dead eggs at 1, 2, and 4 WPT and in the mean percentage of mature eggs at 2 and 4 WPT compared with the nontreated infected mice. At 4 WPT, more than 50% of the eggs were mature. As the female worm starts oviposition 30 days after infection and the laid immature eggs take about 6 days to mature, a reduction in the number of immature eggs and/or increase in mature or dead eggs indicate that the chemical examined interfered with oviposition. Massoud et al. [15] attributed the changes in the oogram pattern produced by MZD to an early interruption of egg laying in the intestinal wall or the blocking of oviposition development. Also, Badria et al. [24] reported that MZD caused separation of male and female-coupled worms and shifting of female worms from their normal habitat to the liver resulting in progressive reduction of immature eggs deposited in the wall of the small intestine with an increase in mature eggs (93%), but they did not report on dead eggs. These results do not coincide with those of other studies [1],[27],[28], which found an absence of oogram alterations after MZD treatment of mice or hamsters.


  Conclusion Top


MZD showed moderate (72%) antischistosomal activity in the animal model at the assigned dose after treatment.

Acknowledgements

Many thanks to Dr Rashad Abdulghani, Department of Parasitology, Faculty of Medicine and Health Sciences, Sana’a University, Sana’a, Yemen, for his support in the practical part of this work.

Author Contribution

MA Al Kazzaz suggested the idea of this work, searched the literature, statistically analyzed the data, and prepared the manuscript; MH El-Sayad supervised the study and designed the experiments and shared in the revision of the manuscript; SA Abu Helw shared in the supervision of the study, data analysis, and revision of the manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]


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