Parasitologists United Journal

Decem

ber 2014 Volum

e 7 • Num

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Antischistosomal activity of Mirazid in experimentalschistosomiasis mansoni: exploring the controversyMohammad A. Al-Kazzaz, Mona H. El-Sayad, Sahar A. Abu-Helw

Department of Parasitology, Medical Research

Institute, Alexandria University, Alexandria,

Egypt

Correspondence to Mohammad A. Al-kazzaz,

MSc, 27 Street, 15 Al-Awayed, Alexandria,

Egypt Tel: + +20 100 119 5791;

e-mail: mohammadaziz73@gmail.com

Received 14 December 2015

Accepted 22 March 2016

Parasitologists United Journal2016, 9:31–36

BackgroundMirazid (MZD) was licensed in Egypt for treatment of schistosomiasis in the year2002; the drug gained much attention experimentally and clinically, with conflictingviews on its efficacy.ObjectiveThe study aimed to evaluate MZD anti-schistosomal activity in an animal model at aselected dose.Materials and methodsSwiss albino mice (n=36) were infected withSchistosomamansoni and divided intotwo equal groups of 18 mice each: group 1 was the nontreated infected controlgroup given only the vehicle; gourp 2was infected and treated withMZD at a dose of500mg/kg for 5 days per os 7 weeks postinfection. Efficacy of the drug wasassessed parasitologically with fecal egg counts evaluated every other day untilthe animal was euthanized at 1, 2, and 4 weeks post-treatment (WPT); wormburden, tissue egg count and oogram pattern were studied at 1, 2, and 4 WPT.ResultsMZD reduced fecal egg counts in infected mice (69.6%) and reduced total wormload (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 oftreatment.ConclusionMZD has moderate antischistosomal activity in animal models.

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

Parasitologists United Journal 9:31–36

© 2016 Egyptian Parasitologists United Society

1687-7942

IntroductionMirazid (MZD) is a natural pharmaceuticalpreparation introduced into the Egyptian market byPharco Pharmaceuticals (Alexandria, Egypt) in theform of soft gelatin capsules since 2002. Eachcapsule contains 300mg of the purified oleoresinextract of Commiphora molmol (Myrrh) [1]. TheEgyptian drug authority in the Egyptian Ministry ofHealth registered this product for treatment ofschistosomiasis (Reg. No. 21655/2002) [2]. Theantischistosomal properties of MZD wereinvestigated, and reviewed both experimentally andclinically with controversy regarding its efficacy [3].

In humans, the efficacy of MZD as an anti-schistosomal drug was evaluated clinically withpositive results [4–9]. All in all, they enrolled 365schistosomiasis patients (adults and children) andtreated them with MZD at a dose of 10–11.5mg/kgfor 3–6 days. The cure rate, evaluated parasitologically(disappearance of fecal eggs) by Kato–Katz and rectalsnip techniques at 2–3 months after treatment, was80.7–100%. The drug proved to have nonsignificantside effects. The drug was tested by other researchers,

with disappointing results. It showed little or nobeneficial activity as reported by Barakat et al. [10],Botros et al. [11], and Osman et al. [12], whoaltogether enrolled 206 patients (adults or children)treated with MZD at a dose of 300–600mg daily for3–6 days. The drug showed a cure rate that ranged from3.7 to 15.6 % at 3–8 WPT.

Such controversy prompted us to study the efficacy ofMZD experimentally as there are many parasitologicalparameters other than fecal egg counts.

Materials and methodsExperimental studyThe study was conducted at the Department ofParasitology, Medical Research Institute (MRI),Alexandria University, Alexandria, Egypt, during theperiod from 21 July 2013 to 14 October 2014.

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Original article 31

© 2016 Parasitologists United Journal | Published by Wolters Kluwer - Medknow DOI: 10.4103/1687-7942.192993

MaterialsThe study included 36, 8-week-old female Swiss albinomice of the CD-1 strain weighing 20±2 g, obtained fromthe animal house of the MRI, Alexandria University,Alexandria, Egypt. Laboratory-bred Biomphalaria

alexandrina snails infected with miracidiae of theEgyptian (CD) strain of Schistosoma mansoni wereobtained from the Schistosome Biologic Supply Center,TheodoreBilharzResearchInstitute,Cairo,Egypt.MZDcapsules were obtained as free medical samples fromPharco Pharmaceuticals, Batch No: 296. Each capsule(300mg) was emptied in a flask containing 3ml of 4%CremophorELusedasthevehiclefordrugadministration.

MethodsCercarial shedding

Infected B. alexandrina snails were washed withdechlorinated water and kept in an aerated aquarium(using an electric pump) in a dark place (by covering theglass bath with a black plastic bag) [13]. Before use, snailswere rinsed gentlywith a small volume ofwater to removefeces and other debris, then resuspended in water (1ml/snail) and left uncovered in a glass test tube under whitefluorescent light for a period of 30–60min to releasecercariae (Fig. 1;1). After shaking gently to ensurehomogenous distribution of cercariae, 1ml of cercarialsuspension was pipetted and placed on glass slides; adrop of iodine was added to each slide to kill and stainthe cercariae (Fig. 1;2). With the aid of a stereobinocularmicroscope, the number of cercariae was counted in eachslide. Generally, three counts were made in 3ml cercarialsuspensionandtheaveragenumberper1mlwascalculated.

Experimental infection

Mice were infected using the paddling techniqueaccording to Smithers and Terry [14]. Each mousewas exposed separately to about 100 S. mansoni

cercariae (Fig. 1;3). Infected mice were thensegregated in separate stainless steel wire meshcages, and received a standard well-balanced dietand water. The mice were housed in a roomunder controlled environmental temperature. Stoolexamination was performed 50 days after cercarialinfection to determine the presence of S. mansoni eggs.

Drug administrationMZD was administered at a dose of 500mg/kg for5 days per os, 7 WPI. Each mouse required 0.1mlsolution to reach a dose of 500mg/kg. The dose wasselected as specified by Botros et al. [1] andMassoud et al. [15] and is four-fold the therapeuticdose in mice (125mg/kg) based on Food and DrugAdministration guidelines by converting the humandose to those for experimental animals.

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

Figure 1

Steps of mice infections: (1) cercarial shedding; (2) cercarial count;(3) cercarial inoculation.

32 Parasitologists United Journal 2016, Vol. 9 No. 1

Statistical analysisThe data were coded, collected, tabulated, and analyzedusing the independent two-sample t-test with Minitabstatistical software, version 14 (Minitab Inc.,Pennsylvania State College, Pennsylvania, USA).Descriptive statistics were expressed as arithmeticmean ±SD as measures of central tendency anddispersion, respectively. The level of significance(P<0.05) was considered statistically significant. Thechange% in each parasitological parameter wascalculated according to the following equation.

Ethical considerationsThe study protocol was reviewed and approved by theEthicsCommitteeof theMRI,UniversityofAlexandria.

ResultsMZD resulted in a nonsignificant reduction in thenumber of S. mansoni eggs in the stool of the treatedgroup (4.5%) 1WPT followed by significant reduction of39.5 and 69.6% at 2 and 4WPT, respectively, comparedwith the nontreated infected group (Fig. 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, malesbeing more affected than females at 1 and 2 WPT withsignificant reduction of 37.9 and 56.7% compared with25.3 and 36.8%, respectively. At 4 WPT, there wassignificant reduction of both male (75.1%) and femaleworms (60%) (Table 1).

MZD produced significant reduction in the tissue eggcounts in the intestine (29.1–66%) throughout thefollow-up weeks. Reduction in the hepatic tissue eggload was nonsignificant at 1WPT (22.1%), and becamesignificant in the second and fourth week (42.7 and

Figure 2

Egg counts in the stool of MZD-treated Schistosoma mansoni-infectedmice compared with nontreated infectedmice. MZD,mirazid.

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

Group WPT Fecal egg counts Total worms Total males Total females

Group 1: nontreated 1 566.00±55.05 19.60±4.51 12.9±0.55 6.70±1.00

2 765.00±70.83 28.25±5.19 18.50±1.50 9.75±1.50

4 720.00±62.11 28.50±3.54 18.50±0.70 10.00±0.71

Group 2: mirazid treated 1 540.00±42.74(4.5) 13.00±2.71a(34) 8.00±1.53a(37.9) 5.00±0.58a(25.3)

2 462.50±39.09a(39.6) 14.16±2.63a(50) 8.00±1.00a(56.7) 6.16±0.58a(36.8)

4 218.75±12.97a(69.6) 8.00±3.79a(72) 4.60±1.53a(75.1) 3.40±1.15a(66)

Values were expressed as mean±SD; numbers in parentheses indicate the percentage of reduction compared with the infectednontreated group. WPT, weeks post-treatment. aStatistically significant at P<0.05.

Change in treated ð%Þ ¼ mean values in nontreated ðcÞ �mea n values in treated ðtÞmean values in nontreated ðcÞ × 100

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

Group WPT Tissue egg counts Oogram pattern

Intestine Liver Immature Mature Dead

Group 1: nontreated 1 10.3±1.13 4.56±2.84 59.60±2.28 32.60±1.99 7.80±0.59

2 14.6±1.23 6.29±3.59 61.50±3.00 27.00±0.16 11.50±0.38

4 16.5±2.76 7.82±5.80 62.50±0.71 24.50±0.12 12.00±0.41

Group 2: mirazid treated 1 7.30±1.3a(29.1) 3.55±1.28(22.1) 55.50±4.42 19.37±2.16a 24.83±2.62a

2 7.46±0.5a(48.9) 3.60±0.22a(42.7) 23.25±0.99a 39.75±1.71a 37.00±2.55a

4 5.62±0.57a(66) 1.76±0.20a(77.4) 10.77±0.52a 52.00±3.00a 37.33±2.52a

Tissue egg count (EPG)×103 (%R). EPG, eggs per gram; WPT, weeks post-treatment. aStatistically significant at P<0.05.

Antischistosomal activity of mirazid Al Kazzaz et al. 33

77.4%) (Table 2). In the treated group the percentage ofdeadeggs at 1, 2, and4WPTand themeanpercentage ofmature eggs at 2 and 4WPTwere significantly increasedcomparedwith thevalues innontreated infectedmice.At4WPT, more than 50% of the eggs were mature, whichdenotes interruption in egg laying as the number ofimmature eggs decreased with increase in the numberof mature and dead eggs.

DiscussionMZD was tested in in vitro studies by Hassan et al.[19], Sharaf [20], and Bakr et al. [21] in whichthey exposed S. mansoni worms to variousconcentrations of MZD from 100 to 400 μg/ml.The drug elicited maximal somatic musclecontraction at the highest concentration and astrong lethal effect occurred after 24 h exposure.Karamustafa et al. [22] verified the lethal effects ofMZD on adult worms and larvae of S. mansoni

(IC50=7.18–32.69 μg/ml).

In our in vivo study, MZD was tested experimentallyin S. mansoni-infected mice at a selected dose of500mg/kg for 5 days, producing a significantgradual decrease in the total number of worms up to72% by the fourth WPT but did not abolish theinfection. Using the same dose for 5 days, Massoudet al. [15] had reported a 98% reduction in total wormload 45 days post-infection, whereas Botros et al. [1]found that the drug showed only 9% total wormreduction. Bakr et al. [21] also used the same doseof 500mg/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 of600mg/kg for 3 days led to 81.1% worm reduction27 days PT. Shorter periods of effective response toMZD treatment were reported by Badria et al. [24]who used the drug at a dose of 500mg/kg twice a dayfor 3 days and recorded 75% worm burden reductionafter 5 days PT. Lotfy et al. [25] also used a dose of600mg/kg for 5 days with significant worm loadreduction (69.3%) at 4 WPT. El-Gamal et al. [26]studied the effect of a single dose administered 6 weeksafter infection and the drug eradicated 58.6% of totalworms at 2 WPT. However, contradictory resultsreported [1,27–33] showed low worm burdenreduction rates, not more than 10%, in S. mansoni,S. haematobium, and S. japonicum-infected mice orhamsters treated with oral doses of MZD thatvaried from 250 to 500mg/kg for 2–5 days. Suchcontroversial results are expected because ofvariations in drug dosage and/or number of days ofadministration.

In the present work, the number of male and femaleworms decreased, males being more affected thanfemales at 1 and 2 WPT with significant reductionof 37.9 and 56.7% compared with 25.3 and 36.8%,respectively. At 4 WPT, there was significantreduction in the number of both male and femaleworms (66%). These results apparently conform withthose of Bakr et al. [21] for male worms (75.1%) butnot for female worms (83.8%) from MZD-treatedmice 4 WPT with 500mg/kg for 3 days; but Botroset al. [1] found nonsignificant reduction in male andfemale worms at 2 WPT (12.4 and 24.5%),respectively.

Relative to the decrease in the number of worms thedrug resulted in nonsignificant reduction in thenumber of S. mansoni eggs in the stool (4.5%) at 1WPT, but it caused significant reduction (39.6 and69.6%) at 2 and 4 WPT compared with the non-treated infected group. Another experimental report[34] recorded 6.6% followed by 44% reduction in fecalegg counts at 2 and 4 WPT, respectively, using500mg/kg MZD for 5 days. In contrast, using ahigher dose of 600mg/kg for 6 days, 97% reductionin fecal egg counts was induced after 4 weeks of MZDtherapy [25].

MZD resulted in significant reduction in tissue eggcounts in the intestines (48.9–66%) throughout the 4WPT follow-up in our study. Nonsignificantreduction in the hepatic tissue egg load wasobserved at 1 WPT (22.1%) but the drug was ableto significantly reduce the egg count in the second andfourth WPT (42.7 and 77.4%, respectively). Highertissue egg count reduction of 98.2 and 97% in theintestinal and hepatic tissue egg counts, respectively,was recorded at 5 WPT [15]. In contrast, anotherresearch [21] recorded a lower percentage reduction of41% and 28.9% in hepatic and intestinal tissue eggcounts at 4 WPT and with a similar MZD dose of500mg/kg, but for 3 days. Other studies [1,27] foundnonsignificant change in both intestinal and hepatictissue egg loads at 2 WPT with a dose of MZD 300 or500mg/kg for 3 or 5 days, and examined 7 WPI.Regarding the oogram pattern, MZD resulted insignificant increase in the percentage of dead eggsat 1, 2, and 4 WPT and in the mean percentage ofmature eggs at 2 and 4 WPT compared with thenontreated infected mice. At 4 WPT, more than 50%of the eggs were mature. As the female worm startsoviposition 30 days after infection and the laidimmature eggs take about 6 days to mature, areduction in the number of immature eggs and/orincrease in mature or dead eggs indicate that the

34 Parasitologists United Journal 2016, Vol. 9 No. 1

chemical examined interfered with oviposition.Massoud et al. [15] attributed the changes in theoogram pattern produced by MZD to an earlyinterruption of egg laying in the intestinal wall orthe blocking of oviposition development. Also, Badriaet al. [24] reported that MZD caused separation ofmale and female-coupled worms and shifting offemale worms from their normal habitat to the liverresulting in progressive reduction of immature eggsdeposited in the wall of the small intestine with anincrease in mature eggs (93%), but they did not reporton dead eggs. These results do not coincide with thoseof other studies [1,27,28], which found an absence ofoogram alterations after MZD treatment of mice orhamsters.

ConclusionMZD showed moderate (72%) antischistosomalactivity in the animal model at the assigned doseafter treatment.

AcknowledgementsMany thanks to Dr Rashad Abdulghani, Departmentof Parasitology, Faculty of Medicine and HealthSciences, Sana’a University, Sana’a, Yemen, for hissupport in the practical part of this work.

Author ContributionMA Al Kazzaz suggested the idea of this work,searched the literature, statistically analyzed the data,and prepared themanuscript;MHEl-Sayad supervisedthe study and designed the experiments and shared inthe revision of the manuscript; SA AbuHelw shared inthe supervision of the study, data analysis, and revisionof the manuscript.

Financial support and sponsorshipNil.

Conflicts of interestThere are no conflicts of interest.

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