Mohammad aziz,m sc thesis 2014

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‫ميحرلا نمحرلا هللا‬ ‫بسم‬
‫ارثين‬‫و‬‫ال‬ ‫ونجعلهم‬ ‫ائمة‬ ‫ونجعلهم‬ ‫االرض‬ ‫في‬ ‫ا‬‫و‬‫استضعف‬ ‫الذين‬ ‫علي‬ ‫نمن‬ ‫ان‬ ‫ونريد‬)٥(‫ونمكن‬
‫ن‬‫يحذرو‬ ‫ا‬‫و‬‫كان‬ ‫ما‬ ‫منهم‬ ‫وجنودهما‬ ‫وهامان‬ ‫ن‬‫عو‬‫فر‬ ‫ي‬‫ونر‬ ‫االرض‬ ‫في‬ ‫لهم‬)٦(
‫كريم‬ ‫قرأن‬/‫القصص‬ ‫سورة‬
By
Dr.Mohammad Aziz
Department of Medical Parasitology
Medical Research Institute, Alexandria University
October 14,2014

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Study of the efficacy of Nitazoxanide ,
Myrrh Total Oil and Mirazid in comparison
with Praziquantel in experimental
Schistosomiasis mansoni
--------------------------------------------------------

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Schistosomiasis is a parasitic disease
caused by the digenetic trematodes of
the genus Schistosoma (blood flukes).
The disease is one of ten tropical
diseases especially targeted for
prevention and control by the special
programs for research in WHO.

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Etiology of schistosomiasis
• Schistosoma mansoni S.hematobium
• S.japonicum
• S.intercalatum
• S.mekongi
There are some animal or bird schistosome
species may infect human .
UrinaryIntestinal

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Oviposition commences 4-7 weeks post infection
adult worm passing eggs
egg into fresh water
cercariae
miracidia
penetrate into the body of the snail
(intermediate host)
Schistosomes are characterized by a complex life cycle involving two phases;
1-sexual phase in which sexual reproduction by adult worms in humans (definitive host),
2-asexual phase in specific aquatic snails (intermediate host,Biomphalaria species).
Life cycle and Biology of S.mansoni

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EPIDEMIOLOGY
OF
SCHISTOSOMIASIS MANSONI

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 Source of infection: infected humans.
 Mode of infection : skin penetration by
cercariae .
 three major factors are responsible for the
occurrence of schistosomiasis:
1. The method of disposal of human excreta
2. The presence of the snail intermediate host
3. The contact of human with cercaria-infested
water.

9
B.alexandrina has historically been implicated
in the transmission of S.mansoni in Egypt.

10
In 2012 ,at least 249 million people required
preventive treatment for Schistosomiasis,
only 42 million have been treated (WHO).
on revising the global burden of
schistosomiasis ; there was about 280,000
deaths per year in sub-Saharan Africa alone
where 130,000 per year due to hematemesis
from S. mansoni .

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• Fenwick (2011) reported that prevalence for
Schistosomiasis in allover the country was
less than 0.5 % .
• WHO (2011) found only 20 villages in the
whole country had prevalence (3-9%).
• But higher prevalence rates than previously
reported were present in some areas in Egypt
as in Kafr El-Sheikh Governorate , the
prevalence rate was (16 %) among School-
children (Khalil 2013).
• Taman et al., (2014) found the prevalence rate
(26%) in a survey on fishermen in Al-Manzala
lake.

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• The main immunopathology of the disease is
granuloma formation in the liver and other tissues.
• Chronic schitosomiasis is manifested by periportal
fibrosis) .
 Hepatosplenomegaly .
 Portal hypertension.
 Esophageal varices .
 Pulmonary hypertension
 Cor pulmonale
 Neuroschistosomiasis
 Infertility
Pathological Aspects of Schistosomiasis Mansoni
Complications of Schistosomiasis Mansoni

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Chemotherapy of
Schistosomiasis

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The Current Antischistosomal Therapy of
Schistosomiasis Mansoni depends on:
• Praziquantel is the drug of choice which is
available all-over the world since 1980 .
• Mirazid is present only in Egypt since 2002 .
The aim of chemotherapy is to reduce
the morbidity in the infected human

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• The antiparasitic activity of PZQ was observed in the early
1970s at the laboratories of Bayer and E.Merck, Germany.
• PZQ has been investigated, both experimentally and
clinically against schistosomiasis with higher degrees of
trustability regarding its efficacy .

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Advantages of praziquantel in treatment of
schistosomiasis:
PZQ is characterized by
- high efficacy,
- excellent tolerability,
- few and transient side effects,
- simple administration,
- competitive cost.
- The drug is equally suited for individual or
large scale treatment .
So PZQ deserves to be included in the WHO
model list of essential drugs .

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-----------------------------------------------
Mirazid is a pharmaceutical natural
preparation from purified oleoresin extract of
Commiphora molmol (Myrrh).
It introduced to the Egyptian market in the
form of soft gelatin capsules by Pharco
pharmaceuticals .
Each capsule contains 300 mg.
(Reg.No.21655/2002).

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-MZD has been launched as a safe drug of natural
origin.
-The drug has been investigated, both experimentally
and clinically against schistosomiasis with controversy
regarding its efficacy .

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* The fear for possible emergence of drug
tolerance or appearance of new resistant
strains to PZQ especially with reinfection and
re-treatment makes the search for new
antischistosomal drugs an essential target
* The process of Drug Discovery and
Development of novel antischistosomal agents
either chemically designed or naturally is
continously performed.
Novel Treatments of Schistosomiasis
under research

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Synthetic Natural
1.PZQ derivatives 1.Artemisia
2.Oxadiazoles (Furoxan derivatives) 2.Myrrh eg. Myrrh oil
3.Cysteine Protease Inhibitors 3.Citrus reticulate
4.Trioxaquines 4.Ailanthus altissima
5.Trioxolanes (secondary ozonides) 5.Curcumin (Curcuma longa)
6.Imidazolidines 6.Ginger (Zingiber officinale)
7.Benzimidazole derivatives 7.Agave Americana var. marginata
8.Thiazoles eg.Nitazoxanide (NTZ) 8.Nigella sativa
9.Oxamniquine derivatives 9.Garlic (Allium sativum)
10.Thiazolo-Derivatives 10.Pinus canariensis
11.Nano-compounds 11.Euphorbia schimperiana
12.Adenine derivatives 12.Balanites aegyptiaca
13.Benzothiazoles 13.Sesbania sesban
14.Nucleoside phosphonates 14.Conyza dioscorides
15.Substituted Pyrimidinedione derivatives 15.Chenopodium ambrosioides
16.Thioxo-imidazolidine compounds 16.Ferula assafoetida
17.Benzodiazepines 17.Ziziphus spina christi
18.Aryl Ozonides 18.Cleome droserifolia
19.Anti-androgens 19.piplartine (Piper)
20.Miscellaneous group 20.Holothuria polii
1. Antox 21.propolis
2. pegylated tartar emetic 22.Furcraea selloa
3. Ro 15-5458 23.Spirulina platensis
4. Ro-354 24.Carica Papaya
5. Clorsulon 25.Pomegranate (Punica granatum)
6. Ozone 26.Eugenia edulis
7. Mefloquine 27.Brachychiton rupestris
8. Arachidonic acid 28.Solanum nigrum
9. Interferon 29.Solanum elaeagnifolium
10. Miltefosine 30.Callistemon viminalis
11. endoperoxide N-89 31.Verbascum sinaiticum
12. Licarin 32.Eucalyptus citriodora
13. Ivermectin 33.Asclepias sinaica
14. Imatinib 34.Calotropis procera
35.Pergularia tomentosa
36.Alkanna orientalis
37.Khaya grandifoliola
38.Swietenia mahogany
39.Pimenta racemosa
40.Morus alba
Nitazoxanide
Myrrh Total Oil

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A considerable number of these agents
were tested and proved promising anti-
schistosomal activities , the majority of
them were consigned to the museums of
history, but few succeeded in reaching
more advanced developmental phases of
clinical trials without reaching the
consumer in the real life .

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 Experimental S.mansoni infection of
laboratory animals has frequently been used
to study the anatomical, pathological and
physiological features of schistosomiasis in
humans as well as for the study of immunity
and chemotherapy .
 Mice have tended to be the animals of choice
because of their easy availability, high
fertility and susceptibility to experimental
infection.

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AIM OF THE WORK
The aim of the study is to assess efficacy of
Nitazoxanide, Myrrh Total Oil and Mirazid
in comparison with Praziquantel in
treatment of S.mansoni -infected mice.

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120 mice were randomly allocated
through infected groups (100 mice,G1-G5)
and non-infected group (20 mice,G6).

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Mice infection
• infected B.alexandrina snails
were obtained from TBRI.
• snails were exposed to white
fluorescent light for a period of
30-60 min to release cercariae
(shedding).
 Each mouse was exposed
separately to about 100
S.mansoni cercariae by
paddling technique.
• 100 mice were infected and
Hygeinically housed .

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 Stool examination was performed 50 days
post-cercarial infection to investigate the
presence of S.mansoni eggs.

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Group1: infected and treated orally with MZD 500
mg/kg bw/day for 5 consecutive days .
Group 2: infected and treated orally with MTO 18 mg
/kg bw/day for 3 consecutive days .
Group 3: infected and treated orally with NTZ 100
mg/kg bw /day for 7 consecutive days .
Group 4: infected and treated orally with PZQ 500
mg/kg bw /day for 2 consecutive days .
Group 5: infected and non-treated (+control G).
Group 6: normal non-infected and non-treated
(-control G).
Mice were sacrificed at 1, 2 and 4 weeks post-treatment

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Evaluation of drug efficacy was based on the following parameters :
I.Parasitological Studies :
a-Fecal egg counts (eggs were counted every other day starting
2 days post-treatment and continued till mice sacrifice ).
b-Worm burdens, sexes and lengths
c-Tissue egg counts (liver and intestine)
d-Oogram patterns.
2.Scanning Electron Microscpic Study:
3.Hematological Studies :(CBC).
4.Biochemical studies:
- liver functions tests (ALT,AST and ALP) .
- kidney functions tests (urea and creatinine)
- Cholinesterase level

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The % of change between treated and non-treated
groups was calculated as follow:
% change in treated =
Mean values in non-treated(c) - Mean values in treated (t) × 100
Mean values in non-treated (c)

35
Egg counts in stool of S. mansoni-infected mice under different
treatments compared to non-treated infected mice.
0
100
200
300
400
500
600
700
800
900
3 5 7 9 11 14 16 18 20 22 24 26 28
MeanEggCountsinstool(epg)
Days of Follow Up
NTZ MTO MZD PZQ non-treated

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Percentage faecal egg count reduction in S. mansoni-infected mice under
different treatments compared to non-treated infected mice .
0
20
40
60
80
100
NTZ MTO MZD PZQ
4.9
1
4.5
63.9
22.5
9.8
39.5
100
50.6
12.5
69.6
100
%FaecalEggCountReduction
Mice Groups
1w 2w 4w

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Groups
WPT
Total worm
Burden
(Mean ± SD)
TWR
%
NTZ
1 14.60±1.81 26
2 15.75±0.95 A 45
4 10.00±1.82 A 65
MTO
1 18.00±4.83 9
2 20.67±3.21 27
4 20.33±2.08a 29
MZD
1 13.00±2.53 a 34
2 14.25±2.63 A 50
4 8.25±1.50 A 71
PZQ
1 3.33±1.75 A 83
2 1.6±0.54 A 94
4 1.00±0.70 A 97
Infected
non-treated
1 19.80±2.86 -
2 28.67±5.51 -
4 28.67±4.73 -
Effect of different drugs
on Total worm burden
reduction in S.mansoni-
infected mice .
WPT: weeks post-treatment,
TWR:Total worm reduction,
NTZ = Nitazoxanide,
MTO= Myrrh total oil,
MZD=Mirazid,
PZQ= praziquantel,
Values were expressed as
mean ± SD.
a: Statistically significant at
P.value < 0.05.
A : Statistically highly
significant at P.value < 0.01.

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The tissue egg counts in the liver and intestine of S.
mansoni-infected mice under different treatments at
different periods of follow up.
Group
Tissue egg count(epg) x103 (% R)
Intestine Liver
1st week 2nd week 4th week 1st week 2nd week 4th week
NTZ 8.00±1.1A
(-22%)
8.4±1.6A
(-45%)
8.8±0.7A
(-47 %)
3.6±1.4
(-20%)
4.8±0.8a
(-24%)
5.4±0.5A
(-31%)
MTO 9.02±1.06a
(-12%)
11.2±0.7A
(-23%)
11.3±1.7A
(-31%)
4.15±1.18
(-7%)
4.66±1.4
(-26%)
4.5±1.9a
(-43%)
MZD 7.30±1.3A
(-29%)
7.4±0.5A
(-49%)
5.6±1.00A
(-66%)
3.5±1.6
(-22%)
3.6±0.8A
(-43%)
2.7±1.0A
(-65%)
PZQ 3.09±0.16A
(-70%)
3.05±0.9A
(-79%)
1.96±0.4A
(-88%)
1.6±0.7A
(-64%)
1.94±0.8A
(-70%)
1.1±0.19A
(-89%)
Infected
Non-treated 10.28±0.4 14.6±0.3 16.5±0.6 4.5±0.7 6.3±0.6 7.8±1.3
% R: Percent of reduction,WPT: weeks post-treatment, NTZ = Nitazoxanide,MTO=Myrrh total oil,
MZD= Mirazid, PZQ= praziquantel,Values were expressed as mean ± SD. a: Statistically
significant at P value < 0.05., A : Statistically highly significant at P value < 0.01.

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Mean Percentage of egg developmental changes in S.mansoni-infected mice
under different treatments at different periods of follow up.
NTZ MTO MZD PZQ Non-treated
1w 19.5 16.5 24.83 79.87 7.8
2w 22.25 20 37 83.2 11.5
4w 30.5 26.5 37.33 85.75 12
0
10
20
30
40
50
60
70
80
90
100mean%ofdeadeggs
1w 2w 4w
0
10
20
30
40
50
60
mean%ofmatureeggs
1w 2w 4w
0
10
20
30
40
50
60
70
mean%ofimmatureeggs
1w 2w 4w
1
3
2

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II. Scanning electron microscopic study

41
• PZQ showed a pronounced tegumental damage in the form
of rupture of tubercles and loss of spines in wide areas in
male worms.
• Some teguments showed severe erosion or even sloughing of
tegumental membranes exposing the underlying muscle
layers.
Scanning electron micrographs of the tegument of male S.mansoni worms
recovered from infected non-treated mice (A) and PZQ-treated worms (B).
ITR
T
S
T
ITR S

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* Marked ulceration in the tegument was detected in the
outer surface of female worms after PZQ treatment .
Scanning electron micrographs of the tegument of female S.mansoni
worms recovered from infected non-treated mice (C) and PZQ-
treated worms (D).
S
U

43
• MZD showed mild tegumental damage in male S. mansoni worms
in the form of rupture of tubercles with mild loss of spines and if
present, lost their sharpness .
• There was no obvious deeper effects in the teguments as the
changes were topically confined to the outer surface.
recovered from infected non-treated mice (A) and MZD-treated worms (B).
S
T
ITR
T
ITR
S

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• in the female tegument, There was focal erosion and
ulceration with shrinkage of the outer surface after MZD .
• There was higher sensitivity in the tegumental damages in
males than females.
Scanning electron micrographs of the tegument of female S.mansoni
worms recovered from infected non-treated mice (C) and MZD-treated
worms (D).
S
U

45
• The ventral sucker still intact in MZD-treated
worms.
Scanning electron micrographs of ventral sucker of S.mansoni worms recovered
from infected non-treated mice (E) and MZD-treated mice (F).
VS
VS

46
recovered from infected non-treated mice (A) and NTZ-treated worms (B).
* NTZ resulted in mild tegumental damaging effect
manifested only by focal lesions in the inter-tubercular
ridges of male worms.
T
ITRS
T
S
ITR

47
Scanning electron micrographs of the tegument of female S.mansoni worms recovered
from infected non-treated mice (C) and NTZ-treated worms (D).
No effect of NTZ on the tegument of female worms.
s
s

48
• disorganization of the oral suckers of male
worms under NTZ -treatment.
Scanning electron micrographs of oral sucker of S.mansoni worms recovered
from infected non-treated mice (E) and NTZ-treated mice (F).
os
os

49
• loss of spines in the gynecophoric canal in
NTZ-treated worms.
Scanning electron micrographs of the the gynecophoric canal
of S.mansoni worms recovered from infected non-treated mice
(G) and NTZ-treated mice (H).
s

50
• No effect of MTO on the male teguments
recovered from infected non-treated mice (A) and MTO-treated worms (B).
S
T
ITR
T
S
ITR

51
MTO resulted in oedematous swelling of both oral
and ventral suckers.
Scanning electron micrographs of the oral sucker of male S.mansoni worms
recovered from infected non-treated mice (C) and MTO-treated worms (D).
OS
OS
VS

52
III.Haematological Studies Studies

53
WPI
Infected
Non-treated
Non-infected
Non-treated
8 9.20±0.77A
[-27.5%]
12.70±0.57
9 7.07±0.90A
[-46.6%]
13.26±1.00
11 5.73±0.36A
[-59.4%]
14.12±1.69
HB level in S. mansoni-infected mice at
different follow up periods.

54
WPT PZQ
Infected
Non-treated
Non-infected
Non-treated
1 10.30±0.44b
(+11.9%)
9.20±0.77A
[-27.5%]
12.70±0.57
2 11.10±0.61B
(+57%)
7.07±0.90A
[-46.6%]
13.26±1.00
4 11.90±0.61B
(+107.6%)
5.73±0.36A
[-59.4%]
14.12±1.69
Effect of PZQ on HB level in S.mansoni-infected mice at

55
Effect of MZD on HB level in S.mansoni-infected mice
Effect of MZD on HB level in S.mansoni-infected mice
at different follow up periods.
WPT MZD
Infected
Non-treated
Non-infected
Non-treated
1 9.54±0.35
(+3.6%)
9.20±0.77A
[-27.5%]
12.70±0.57
2 10.25±1.0B
(+44.9%)
7.07±0.90A
[-46.6%]
13.26±1.00
4 11.37±1.11B
(+98.4%)
5.73±0.36A
[-59.4%]
14.12±1.69

56
WPT NTZ
Infected
Non-treated
Non-infected
Non-treated
1 9.70±0.95
(+5.4%)
9.20±0.77A
[-27.5%]
12.70±0.57
2 9.10±0.28B
(+28.7%)
7.07±0.90A
[-46.6%]
13.26±1.00
4 9.40±0.56B
(+64%)
5.73±0.36A
[-59.4%]
14.12±1.69
Effect of NTZ on HB level in S.mansoni-infected mice at

57
WPT MTO
Infected
Non-treated
Non-infected
Non-treated
1 9.70±0.95
(+5.4%)
9.20±0.77A
[-27.5%]
12.70±0.57
2 9.10±0.28B
(+28.7%)
7.07±0.90A
[-46.6%]
13.26±1.00
4 9.40±0.56B
(+64%)
5.73±0.36A
[-59.4%]
14.12±1.69
Effect of MTO on HB level in S.mansoni-infected mice
at different follow up periods.

59
% change in the total leucocytic counts in S.
mansoni-infected mice under PZQ treatment at different
follow up periods.
-60
-40
-20
0
20
40
60
80
100
120
PZQ Non-treated
-10.8
20.7
-17.8
46.9
-48.8
101.2
%ChangeinWBCscount
1w 2w 4w

60
mansoni-infected mice under MZD treatment at
-40
-20
0
20
40
60
80
100
120
MZD Non-treated
0.1
20.7
-14.2
46.9
-35.1
101.2
%ChangeinWBCscount
1w 2w 4w

61
mansoni-infected mice under NTZ treatment at different
follow up periods.
-40
-20
0
20
40
60
80
100
120
NTZ Non-treated
1.2
20.7
-6.6
46.9
-22.5
101.2
%ChangeinWBCscount
1w 2w 4w

62
% change in the tota leucocytic counts in S.
mansoni-infected mice under MTO treatment at
-20
0
20
40
60
80
100
120
MTO Non-treated
2.6
20.7
-2.3
46.9
-9.4
101.2
%ChangeinWBCscount
1w 2w 4w

65
ALT activity in S. mansoni-infected mice treated with
different drugs at different times.
WPT NTZ MTO MZD PZQ Infected
Non-Treated
Non-Infected
Non-Treated
1 53.67±3.11B
(-16.6%)
71.0±5.49b
(+10.2%)
49.25±4.6B
(-23.5%)
45.60±1.4B
(-29.1%)
64.40±3.9A
(54.5%)
41.37±6.21
2 68.00±4.4B
(-26.4%)
93.00±6.6
(+0.5%)
68.50±8.6B
(-25.9%)
60.00±6.5B
(-35.1%)
92.50±3.54 A
(80.6%)
51.20±7.96
4 69.50±7.0B
(-30.7%)
85.00±5.9B
(-15.2%)
60.00±6.7B
(-40.1%)
53.75±6.2B
(-53.5%)
100.33±6.5A
(202.2%)
33.20±5.89

67
Serum creatinine level in S.mansoni-infected mice under
different treatments at different follow up periods.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0.78
1.28 1.3
1.59
1.06
0.90.9
1.18
1.25
1.4
1.25
0.7
0.75
0.9
1.1
1.3
1.45
0.58
MeanSerumCreatininelevel(mg/dl)
1w 2w 4w

69
Blood acetylcholinesterase levels in S.mansoni-infected mice
under different treatments at different periods of follow up.
0
2
4
6
8
10
12
PZQ MZD NTZ MTO Non-treated Non-infected
9.32
9.93
8.6
8.1
9
10.15
9.8
9.3
9.05
7.5
8
9.989.98
9.5
8.8
7.25
7.57
9.9
BloodAcetylcholinesteraselevel(u/ml)
1w 2w 4w

70
Conclusion:
This study declared that PZQ is still the most important drug in
treatment of schistosomiasis because of its high lethality to
schistosome worms as early as possible after two weeks of
treatment with higher safety margins on blood cells, liver and
kidney functions tests as well as blood AChE activity.
MZD was less effective than PZQ in its antischistosomal activity
but highly safe without adverse haemtological or biochemical
effects on infected treated mice.
NTZ was less effective than PZQ and MZD but with less adverse
health effects .
MTO exerted little antischistosomal activity with lower safety
profile at the selected dose.

71
RECOMMENDATIONS
1. Continuity of use of Praziquantel as a standard treatment of
schistosomiasis as the drug is still effective and safe until
production of new antischistosomal agents or vaccines.
2. The haematological profile of PZQ should be re-evaluated as
there is scarcely available literature in this concern.
3. When Mirazid is used as alternative to PZQ for treatment of
S.mansoni infection; adequate doses should be used and
thorough parasitological re-assessment is essential as egg
excretion may continue at a low level.
4. Mirazid and Myrrh total oil are very complex mixture of
compounds so fractionation of them into fine components may
yeild very promising new antischistosomal agents than the very
simple preparation of MZD.

72
5. Short course of treatment in MZD application as in PZQ should
be tested to offer maximum patient compliance.
6. Re-evaluation of cholinesterase activity of MZD in vitro on adult
schistosomes may explore the mechanism of action of the drug.
7. Re-evaluation of NTZ safety in various healthy animal models
with various doses and courses as well as its efficacy in
treatment of schistosomiasis using in vitro and animal models
alone or in combination with PZQ.
8. For experimental discovery of antischistosomal activity of a
substance, adopt WHO criteria to save time and costs, so many
substances may be assessed.

Mohammad aziz,m sc thesis 2014

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Mohammad aziz,m sc thesis 2014