mohammad aziz,m sc thesis 2014

1

بسم ميحرلا نمحرلا هللاونمكن )٥(ونريد ان نمن علي الذين استضعفوا في االرض ونجعلهم ائمة ونجعلهم الوارثين )٦ (لهم في االرض ونري فرعون وهامان وجنودهما منهم ما كانوا يحذرون

سورة القصص / قرأن كريم

By Dr.Mohammad Aziz

Department of Medical Parasitology Medical Research Institute, Alexandria University

October 14,2014

2

Study of the efficacy of Nitazoxanide ,

Myrrh Total Oil and Mirazid in comparison

with Praziquantel in experimental

Schistosomiasis mansoni

--------------------------------------------------------

3

INTRODUCTION

4

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.

5

Etiology of schistosomiasis

• Schistosoma mansoni S. hematobium

• S. japonicum

• S. intercalatum

• S. mekongi

There are some animal or bird schistosome species may infect human .

Urinary Intestinal

6 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

7

EPIDEMIOLOGY

OF SCHISTOSOMIASIS MANSONI

8

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 .

11

In Egypt

12

• 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.

13

• 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

14

Chemotherapy of Schistosomiasis

15

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

16

• 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 .

17

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 .

18

-----------------------------------------------

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).

19

-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 .

20

* 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

21

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

22

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 .

23

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.

24

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.

25

Materials and Methods

26

120 mice were randomly allocated

through infected groups (100 mice,G1-G5)

and non-infected group (20 mice,G6).

27

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 .

28

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

29

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

30

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

31

Mouse perfusion

32

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)

33

RESULTS &

DISCUSSION

34

1-PARASITOLOGICAL STUDIES

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

Mea

n E

gg

Cou

nts

in s

too

l(ep

g)

Days of Follow Up

NTZ MTO MZD PZQ non-treated

36

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

% F

ae

ca

l E

gg

Co

un

t R

ed

uc

tio

n

Mice Groups

1w 2w 4w

37

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.

38

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.

39

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

100m

ean

% o

f d

ead

eg

gs

1w 2w 4w

0

10

20

30

40

50

60


mea

n %

of

mat

ure

eg

gs

1w 2w 4w

0

10

20

30

40

50

60

70


mea

n %

of

imm

atu

re e

gg

s

1w 2w 4w

1

3

2

40

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

42

* 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.

Scanning electron micrographs of the tegument of male S.mansoni worms recovered from infected non-treated mice (A) and MZD-treated worms (B).

S

T

ITR

T

ITR

S

44

• 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

Scanning electron micrographs of the tegument of male S.mansoni worms

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

ITR S

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

Scanning electron micrographs of the tegument of male S.mansoni worms 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 different follow up periods.

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 different follow up periods.

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.

58

2.Leucocytic counts

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

% C

han

ge

in W

BC

s co

unt

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

% C

han

ge

in W

BC

s co

unt

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

% C

ha

ng

e i

n W

BC

s c

ou

nt

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

% C

ha

ng

e i

n W

BC

s c

ou

nt

1w 2w 4w

63

IV-Biochemical studies

64

LIVER FUNCTIONS TESTS

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

66

KIDNEY FUNCTIONS TESTS

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

0.9 0.9

1.18 1.25

1.4

1.25

0.7 0.75

0.9

1.1

1.3

1.45

0.58

Mea

n S

eru

m C

reat

inin

e le

vel

(m

g/d

l)

1w 2w 4w

68

THE ACHE ACTIVITY

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

9.98 9.98 9.5

8.8

7.25 7.57

9.9

Blo

od

Ace

tylc

ho

lines

tera

se l

evel

(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

Health & Medicine