Journal of Thi-Qar University No.2 Vol.

8 March /2013
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In Vitro Study of The Effect of Some Drugs on Leishmania Donavani

 Replication Rate

ϢδΠϟ΍ΝέΎΧΔϴ΋ΎθΣϻ΍ΎϴϧΎϤθϠϟ΍ϲϠϴϔρϮϤϧϝΪόϣϰϠϋΔϴΒτϟ΍ήϴϗΎϘόϟ΍ξόΑήϴΛΎΗΔγ΍έΩ
ϲΤϟ΍
 Ayad Q. Al- Makki
Department of Pharmacology, Toxicology, and Allied Sciences / College of Pharmacy/
 University of Basrah

 Abstract

The effect of some drugs on growth rate of lishmania donovani have been
estimated. The drugs used have different mechanisms of action, some of them tested
on leishmania donavani for the first time. The following drugs used like levamisole in
a dose (1 mg/ml) , metronidazole(1 mg/ml), bromohexine (0.02 mg/ml), sildenafil
(0.5mg/ml) show significant inhibition of L. donavani promastigotes proliferation,
while paracetamole in a dose of (0.1 mg/ml) enhance the promastigotes growth,
meanwhile diclofenac sodium in a dose of (0.5 mg/ml) show little or no effect on
growth rate. Also metronidazole and levamisole in a dose of (0.5 mg/ml) has no effect
on growth rate. The inhibitory effect of drugs appears from the first day of experiment
and fluctuated in second and third days .The effect of paracetamole is continuous
throughout the 3 days of experiment. On the other hand, the effect of diclofenac
sodium unnoticeable till the third day.

Key words: leishmania donavani , levamisole, metronidazole, bromohexine,
sildenafil, paracetamole, diclofenac sodium, in vitro, replication rates

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Journal of Thi-Qar University No.2 Vol.8 March /2013
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Introduction

Leishmaniasis is a disease caused by protozoan parasites that belong to the genus

Leishmania and is transmitted by the bite of certain species of female sand fly (subfamily
phlebotominae)[1]. Most forms of disease are transmissible only from animals, but some can
be spread between humans. They are caused by about 20 species of the protozoan
Leishmania. Leishmania has different types of clinical conditions mostly present as cutaneous
leishmaniasis, another 2 forms are mucocutaneuos & the most severe fatal form is visceral
Leishmaniasis .The treatment of parasitic infections has undergone dramatic changes. The
pantavalent antimonials such as glucantime have been the main anti-leishmaniasis drugs for
over 60 years but their efficacy is diminishing due to drug resistance [2].
Other antileishmanials such as amphotericin B, paromomycin, pentamidin, and
miltefosin are available in cases where antimonials lack efficacy, but their therapeutic
windows are limited .thus, the development of a novel & better anti-leishmanials is
necessary to meet an urgent medical need on a global scale. Immunotherapy with
interferon gamma appears very promising for treatment of visceral Leishmaniasis . In
this study we try to use different types of drugs with different mechanisms of action &
study their effect on proliferation of L. donavani whether enhancing or inhibiting the
parasite growth .drugs used are Levamisole which is antihelmenthic drug produce its
action by inhancing cell mediated immune response probably by action on
macrophage and subsets of T- lymphocyte[3].another drug used in this experiment is
Paracetamole which is selective cyclooxygenase -3 inhibitor that is an enzyme
abundant in brain and spinal cord ,its inhibition cause relief of pain and fever
,Paracetamole has no anti-inflammatory action [4], [5]. Third drug in our study is
Metronidasole which is an antibiotic mainly effective against anaerobic bacteria & is
the drug of choice in treatment of Trichomoniasis. It is subsequently proven to be
effective for both amebiasis and giardiasis [6], and for cutaneous Leishmaniasis [7].
Bromohexine a derivative of plant adhatoda vasica is a potent bronchodilator and
mucolytic ,although it is used for 2000 years in India for treatment of respiratory
ailments and abortifacient activities very few recent reports are available for its
molecular mechanism of action [8]. It has many other properties as antioxidant , anti-
inflammatory , anticestodal and antihelmathic activity [9], [10], [11].
Sildenafil a phosphodiesterase type -5 (PDE5) selective inhibitor that has been
successfully deployed as a drug for treatment of male erectile dysfunction and
pulmonary hypertention[12]. Diclofenac sodium, the sodium salt of O-(2,6-
dichlorophenylamino) ±phenylacetic acid (GP45840, voltarin), is a potant inhibitor of
prostaglandin synthetase. It is suggested that diclofenac Sodium exerts most of its
pharmacological effect via inhibition of prostaglandin synthetase [13].

Aim of study: Find a drug effective against Leishmaniasis so that can be used an
alternative in the management of this disease.

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Materials and Methods

Equipment and Supply

Six drugs have been used as fellow: levamisole: a syrup 40 mg/5 ml kindly
supplied by Kahira Pharm. And Chem. ind. co. Cairo ± Egypt. Paracetamole : 500 mg
tablet supplied by N.D.I. Iraq. Metronidazole : injection 5 mg/ml J. B. Chemicals &
Pharmaceuticals LTD. Ankleshwar ± 393 002, India . Bromohexine : tablets 8 mg
supplied from SDI. , Iraq. Sildenafil : tablet 100 mg from erica life science ltd.
London ,SE6, 4LS, UK. Diclofenac sodium : tablet 50 mg from Troge , Germany. All
drugs were dissolved by sterilized distilled water.

Parasite Isolation
A cloned line of L.donovani (MHOM/IQ/2005/MRC10) was obtained from the
center of medical research at Al-Nahrayn University in Baghdad; this sample is
isolated from the bone marrow of a patient with Kala-azar.

Culture
The promastigotes had been grown on NNN media which is a diphasic media
contain; solid phase [14] and Liquid phase which also called lock solution [15], 2 ml
of lock solution is added to the solid phase of NNN media.

Growth Studies
Growth rate experiment are conducted by inoculating parasites at a density of 1 x
6
10 FHOOPO LQ 111 PHGLD DW  ƕ& DIWHU  KU RI LQRFXODWLRQ 7ZHQW\ VHYHQ
inoculated vials of NNN media has been used for this study with three vials for each
concentration of a given drug. When growth is confirmed after 24 hrs of inoculation
by counting the base line growth rate, the drugs have been added to groups ofVials
each group has three vials and the last group of vials is left as a control media without
addition. The growth rate of parasites at different times (within three consecutive
days) were counted in a Neubauer hematocytometer under light microscopy by using
the following equation:

No. of parasites in 1 mm3 = No. of parasites in five small squares X 800

Data are analyzed with the analysis of variance test and P values < 0.05 are
considered significant.

Results

In the control group, in the second day there is significant difference as compared
to the first day, in the third day there is significant difference as compared to the first
and to the second days. there is gradual increase in growth rates throughout the three
days of experiment. regarding first drug used which is levamisole (Table & Fig 1) , in
group of 1 mg/ml concentration in the first day there is significant difference as

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Journal of Thi-Qar University No.2 Vol.8 March /2013
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compared to the control group . In the second day there is significant difference as
compared to the first day .in the third day there is significant difference as compared
to the second day & as compared to the control group.
Regarding the other group which use 0.5 mg/ml levamisole , in the first day there
is significant difference as compared with control group, and as compared to group of
1 mg/ml levamisole. In the second day the significant difference is found as compared
to the control group and group of 1mg/ml levamisole. While in third day of
experiment the only significant difference is as compared to the second day.
In respect to paracetamole( Table & Fig. 2) we find that in the first day there is
significant difference in comparism to the control group, while in second day there is
significant difference as compared to control group & to the first day as well. In the
third day also there is significant difference as compared to the control group & to the
first day, on the other hand there is no significant difference when compare the second
to the third day.
Regarding metronidazole also there are two doses tested on promastigotes
(0.5mg/ml) and (1mg/ml) (Table & Fig. 3). in the media of growth where 0.5mg/ml
Metronidazole is used, observed that in the first day there is significant difference as
compared to the control group & also to group of 1mg/ml metronidazole. In the
second day , there is significant difference as compared to the first day, to the control
group and to the group of 1mg/ml. in the third day there is significant difference as
compared to the second day & to group of 1mg/ml. On the other hand when use
1mg/ml metronidazole, in the first day there is significant difference as compared to
the control group, while in the second day there is significant difference as compared
to the first day and to the control group, in the third day the significant difference is
found in comparism to the first day and the control group.
Regarding bromohexine 0.02mg/ml (Table & Fig. 4) In the first day, there is
significant difference as compared to the control group, in the second day there is
significant difference as compred to the first day & also a significant difference as
compared to the control group, while in the third day there is a significant difference
as compared to the control group.
In respect to sildenafil ( 0.5mg/ml) ( Table & Fig. 5) In the first day, there is
significant difference as compared to the control group , in the second day there is
significant difference as compared to the first day & to the control group . in the third
day there is significant difference as compared to the second day & to the control
group.
In case of diclofenac sodium( 0.5mg/ml) (Table & Fig. 6) In the first day there is
no significant difference as compared to the control group , in the second day there is
no significant difference as compared to the control group but there is significant
difference as compared to the first day ,while in third day there is significant
difference as compared to control group .

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Table (1): The effects of two concentrations of Levamisole on the L. donovani Promastigote growth rates
in vitro

Levamisole
Count of L. donovani promastigotes expressed in mean+ standard of error
1st day 2nd day 3rd day
MEAN SE MEAN SE MEAN SE
*
CONTROL 5568 + 481.07 11584 + 466.75 19872 + 684.58 *a
Levamisol
2816 + 352.12 b 13120 + 587.88 * 7904 + 406.14 a,b
1mg/ml
b, b,
Levamisol a
7936 + 530.50 c 8992 + 564.33 c 13248 + 682.08
0.5 mg/ml
* Significant different at p<0.05 as compared with first day values
a
Significant different at p<0.05 as compared with second day values
b
Significant different at p<0.05 as compared with controls values
c
Significant different at p<0.05 as compared with 1mg/ml value

CONTROL
25000 Levamisol 1mg/ml
Total No. of Promastigotes/mm3

Levamisol 0.5 mg/ml

20000

15000

10000 19872

13248
5000 11584 13120
7936 8992 7904
5568
2816
0
1st day 2nd day 3rd day
DAYS OF EXPERIMENTS

Figure (1): Illustrate the replication rates of L.donovani promastigotes treated with two
concentration of Levamisol during a period of three days.

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Table (2): The effects of one concentrations of Paracetamole on the L. donovani Promastigote
growth rates in vitro

Paracetamole
Count of L. donovani promastigotes expressed in mean+ standard of error
1st day 2nd day 3rd day
MEAN SE MEAN SE MEAN SE
* *a
CONTROL 5568 + 481.07 11584 + 466.75 19872 + 684.58
Paracetamol ˶ଉb *,b *,b
7808 + 539.19 17728 + 864.94 17056 + 679.95
0.1mg/ml
* Significant different at p<0.05 as compared with first day values
a
Significant different at p<0.05 as compared with second day values
b
Significant different at p<0.05 as compared with control values
c
Significant different at p<0.05 as compared with 1mg/ml values

st nd rd
1 day 2 day 3 day

CONTROL
25000
Total No. of Promastigotes/mm3

Paracetamol 0.1mg/ml
20000

15000

10000 19872
17728

5000 11584
7808
5568
0
1st day 2nd day 3rd d

DAYS OF EXPERIMENTS

Figure (2): Illustrate the replication rates of L.donovani promastigotes treated with (0.1 mg/m
 Paracetamole during a period of three days.

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Metronidazole
Count of L. donovani promastigotes expressed in mean+ standard of error
1st day 2nd day 3rd day
MEAN SE MEAN SE MEAN SE
* *a
CONTROL 5568 + 481 11584 + 467 19872 + 685
Metronidazole ˶ଉb *,b *,b
2656 + 177 9632 + 494 10688 + 482
1mg/ml
Metronidazole 0.5 1040 b, c *,b, c a,c
+ 680 15712 + 694 19744 + 617
mg/ml 0
Table (3): The effects of two concentrations of Metronidazole on the L. donovani Promastigote
growth rates in vitro

* Significant different at p<0.05 as compared with first day values

a
Significant different at p<0.05 as compared with second day values
b
Significant different at p<0.05 as compared with controls values
c
Significant different at p<0.05 as compared with 1mg/ml values

CONTROL

Metronidazole 1mg/ml
Total No.of Promastigotes/mm3

25000
Metronidazole 0.5 mg/ml

20000

15000

10000 19872 19744

15712
5000 10400 11584 10688
st 9632
nd rd
1 day 2 day 3 day
5568
2656
0
1st day 2nd day 3rd day

DAYS OF EXPERIMENTS

Figure (3) : Illustrate the replication rates of L.donovani promastigotes treated with two concentration of Metronidazole
 during a period of three days

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on the L. donovani Promastigote growth

Table (4): The effects of one concentrations of BromohexinBromohexin
vitro Count of L. donovani promastigotes expressed in mean+ standard of error
1st day 2nd day 3rd day
MEAN SE MEAN SE MEAN SE
* *a
CONTROL 5568 + 481 11584 + 467 19872 + 685
Bromohexin ˶ଉb *,b b
1024 + 267 2464 + 206 2176 + 157
0.02mg/ml
* Significant different at p<0.05 as compared with first day values
a
Significant different at p<0.05 as compared with second day values
b
Significant different at p<0.05 as compared with controls values

25000 CONTROL
Total No. of Promastigotes/mm3

Bromohexin 0.02mg/ml
20000

15000

10000 19872

11584
5000
5568
1024 2464 2176
0
1st day
st 2nd day
nd rd3rd day
1 day 2 day 3 day

DAYS OF EXPERIMENTS

Fig (4): Illustrate the replication rates of L.donovani promastigotes treated with one concentration of Bromohexin
 during a period of three days

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Sildenafil
Count of L. donovani promastigotes expressed in mean+ standard of error
1st day 2nd day 3rd day
MEAN SE MEAN SE MEAN SE
* *a
CONTROL 5568 + 481 11584 + 467 19872 + 685
Sildenafil 0.5 ˶ଉb *,b a,b
1760 + 253 7136 + 369 5632 + 290
mg/ml
* Significant different at p<0.05 as compared with first day values
a
Significant different at p<0.05 as compared with second day values
b
Significant different at p<0.05 as compared with controls values

Table (5): The effects of one concentrations of Sildenafil on the L. donovani Promastigote growth rates in vitro

25000 CONTROL
Total No.of promastigotes/mm3

Sildenafil 0.5 mg/ml

20000

15000

10000 19872

5000 11584
5568 7136 5632
1760
0
st nd rd
1st
1 day
day 2nd
2 day
day 3 3rd
dayday
DAYS OF EXPERIMENTS

Figure (5): Illustrate the replication rates of L.donovani promastigotes treated with one concentration of Sildenafil
during a period of three days.

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Table (6): The effects of one concentrations of Diclofenac sodium on the L. donovani Promastigote growth
rates in vitro

Diclofenac
Count of L. donovani promastigotes expressed in mean+ standard of error
1st day 2nd day 3rd day
MEAN SE MEAN SE MEAN SE
* *a
CONTROL 5568 + 481 11584 + 467 19872 + 685
Diclofenac 0.5 5152 + 343 ˶ଉ
10496 + 782 *
10432 + 517 b
mg/ml
* Significant different at p<0.05 as compared with first day values
a
Significant different at p<0.05 as compared with second day values
b
Significant different at p<0.05 as compared with controls values

CONTROL
Diclofenac 0.5 mg/ml
25000
Total No. of promastigotes/mm3

20000

15000

10000 19872

11584 10496 10432

5000
5568 5152

0
1st1stday
day 2nd day
nd
2 day 3rd3day
rd
day
DAYS OF EXPERIMENTS

Figure (6): Illustrate the replication rates of L.donovani promastigotes treated with one concentration of Diclofenac
sodium during a period of three days.

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Journal of Thi-Qar University No.2 Vol.8 March /2013
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Discussion
In the control group there is a gradual increase in growth rate of parasites which
suggest that the media of growth was efficient & well equipped with nutritional
materials so that the large increase in number of parasites not lead to crowding and
competition for survival.
In the present study most types of drugs used have significant difference between
the growth rates of parasites and control group in the first day which mean that they
have killing effect on promastigotes in away or another according to their mechanisms
of action[16]. In the second day most of groups that have been treated with drugs
show an increase in growth rates with a significant difference as compared to the first
day which can be attributed to diminished actin of drugs after 24 hours (there is no
addition) and also to the development of drug resistance[17].Third reason for the
increase in growth rate is that there is a sufficient nutrient & suitable environment in
the media which cause the parasites to replicate again[18].
Regarding Levamisole, 2 doses have been used in the experiment separately (0.5
and 1 mg/ml) 0.5 mg/ml is suboptimal and have no effect on the parasite growth. The
growth rate is larger than that of the control group in the first day and increasing in
the second day and more in the third day. While using 1mg/ml Levamisole leads to
decrease in growth rate in first day with significant difference when compare to
control group. The increase in growth rate in the second day could be explained by
limited duration of action of Levamisole and overgrowth of remaining parasites might
be due to abundant nutrients after large decrease in their numbers in the first day was
also suspected [19].The effect of Levamisole is more apparent in the third day where
it cause large decrease in growth rate and significant difference with control group.
The effect of Levamisole on L.donavani promastigotes is attributed to Levamisole
enhancing effect on cell mediated immunity [3]. Our results goes with a study on
guinea pigs infected with L. donavani, which demonstrate that Levamisole-treated
guinea pigs either did not develop an ulcerative cutaneous lesion or developed a much
smaller lesion than untreated animals[20],the difference from our results is that their
findings studied in vivo.
Regarding Paracetamole , from the data collected we observed that there is a
gradual increase in growth rates in the first & second days more than what had been
calculated in the control group which indicate that the Paracetamole may have
stimulating effect on growth of
L. donavani , the little decrease in growth rate in the third day can be explained by
competition between the parasites on nutritional materials and crowding,our data
could not be explained by the mechanism of action of paracetamole[4].
In case of Metronidazole we observed that the use of small dose 0.5mg/ml leads to
increase in growth rate of parasites even more than the control group in the first &
second day and approximately similar rate of growth between the control group &
group of 0.5mg/ml Metronidazole , this can be explained by development of drug
resistance of parasite when treated by suboptimal dose[17]. While in the other group
where the dose of Metronidazole used is 1mg/ml ,it show significant decrease in
growth rate of parasite which may be an optimal dose.The killing effect of

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Metronidazole appears in the first day more obviously while in second & third day the
growth rate has been increased in comparism to the first day but is less than that of
control group so its effect on parasites may be diminished after 24 hours which may
indicate the necessity of repeating the dose daily . The use of Metronidazole in
treatment of cutaneous Leishmaniasis is proved its efficacy in another study where the
patients lesion had shrunk to half its size by the tenth day of treatment[21].
In regard to Bromohexine the results shows a strongly potent inhibitory action of
Bromohexine on the growth of promasitigotes from the first day & continue to inhibit
growth in the second &third day , There is no significant difference between second
& third days in growth rates but still very low rates, the most accepted explanation for
the present data is that the decrease in growth rates of promasigotes might be related
to Bromohexine inhibiting action on cAMP phosphodiesterase enzymes which is
important for promastigotes proliferation [22].in a study in India prove that peganine
hydrochloride isolated from peganum harmala seeds in dehydrated form is shown to
have exhibited in vitro activity against both extracellular promastigotes as well as
intracellular amastigotes residing within murine macrophage in L. donavani.
Furthermore it also exhibit in-vivo antileishmanial activity[23].
In present study Sildenafil in a dose 0.5mg/ml leads to a significant decrease in
growth rates of promastigotes throughout the three days of experiment, this is because
sildenafil cause inhibition of phosphodiesterase enzyme of L.donavani which shown
to play important roles in cell proliferation & apoptosis of the parasites by controlling
cellular concentration of cAMP and cGMP that are key regulators of many
physiological processes in leishmania[24]. Early studies show that three human
phosphodiesterase enzyme inhibitors (Etazolate, Dipyridamole, and Trequinsin)
inhibit the proliferation of L. major promastigotes and L. infantum amastigotes with
IC50 values in the range of 30 ± 100 µM [25].
The Diclofenac sodium in the experiment show a significant difference in the
second day as compared to the first day but in increasing the Growth not decreasing &
as there is no significant difference as compared to the control group In the second
day so this could be explained as that the Diclofenac sodium has no effect on growth
rate in the second day which mean that the parasite nearly has the same conditions of
Environment as the control group. The only significant difference in decreasing
growth rate was at the third day of experiment when compared to the control group
which mean a delayed action of Diclofenac as mentioned above but still there is a
very little decrease in growth rate from second to third day which may be mean that
the more likely cause of death of parasites is decrease in nutritional supply in addition
to Diclofenac sodium effect and not only to the effect of diclofenac sodium .as the
mechanism of action of Diclofenac sodium is inhibition of prostaglandin E2. It has
EHHQ VKRZQ WKDW DQ LQFUHDVHG SURGXFWLRQ RI 3*( 3*)Į /7& 7;% andPGD2
occur during the courseof murine infection with L.donavani [26]. In a contradictory
report on role of prostaglandin in immunology mention that Prostaglandin of E Series
increase cAMP and by this mechanism it inhibits the Leishmanicidal activity of
macrophage So decrease production of PGs by Diclofenac sodium will cause increase
in the killing of Leishmania by macrophage [27].

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Journal of Thi-Qar University No.2 Vol.8 March /2013
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Conclusion
Selective inhibitors of phosphodiesterase may potentially represent a novel class
of drugs for the treatment of Leishmaniasis .Limited trials of Levamisole on
cutaneous Leishmaniasis may give promising results. Paracetamole can be used in
media for stimulation of Leishmanial growth. being antileishmanial, Bromohexine
can further help to prevent the disease. When use Metronidazole in treatment of
Leishmaniasis, need to give daily dose.

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