subinoculation

Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/2000071043
TRYPANOCIDAL ACTIVITY OF ORGANOTIN CHLORIDES
ON TRYPANOSOMA BRUCEI-INFECTED MICE
S H U A I B U M . N . * , A M E H D A . * * , B O N I R E J . J . * * , A D A U D I A . O . * * * * , I B R A H I M S.** & N O K
A.J.*
Summary:
Résumé
The organotin compounds dibulyltin ( D B T C ) and diphenyltin
INFECTÉES PAR TRYPANOSOMA BRUCEI
dichlorides (DPTC) were tested for trypanocidal activity on a
Les composés de l'organotine,
Trypanosoma
de diphenyltine (DPTC) ont été testés pour leur activité
brucei-infected mice model. At a dose of 1 0 mg
: ACTIVITÉ TRYPANOCIDE DE L'ORGANOTINE CHEZ DES SOURIS
dichlorides
de dibulyltine ( D B T C ) et
D B T C and 1 5 mg D P T C / k g / d a y for five consecutive days, they
sur un modèle de souris
cleared the parasites from the peripheral blood of the infected
doses de 10 mg de DBTC
mice. Subinoculation of some healthy mice with the homogenates
de suite, les parasites
of liver, spleen, kidney, cerebrospinal fluid and blood from the
Après sub-inoculation
mice considered cured, showed a few cases of relapse. The
homogénats de foie, rate, rein, liquide cérébrospinal
L D 5 0 of D B T C and DPTC are 9 0 m g / k g and 7 5 m g / k g
souris considérées
respectively.
été observés.
90 mg/kg
et 15 mg de DPTC/kg/jour
ont disparu du sang des souris
cinq jours
infectées.
de souris en bonne santé avec des
comme guéries,
Les DL50
et 75
trypanocide
infecté par Trypanosoma brucei. Aux
des DBTC
et sang de
quelques cas de rechute ont
et DPTC sont respectivement de
mg/kg.
K E Y W O R D S : organotin, Trypanosoma brucei brucei, trypanocidal.
MOTS CLÉS : organotine, Trypanosoma brucei brucei, trypanocide.
O
rganotins are c o m p o u n d s w h i c h possess o n e
or more direct tin-carbon covalent b o n d ( s )
that are responsible for the specific properties
o f such molecules. T h e c o m p o u n d s assumed c o m mercial significance in the 1970's. T h e y are toxic to a
variety of organisms including bacteria, fungi, protozoa
etc. (Pain & C o o n e y , 1 9 9 8 ) . A large n u m b e r o f organotin c o m p o u n d s show reproducible antitumor activity
in mice (Briddle & Gray, 1 9 8 9 ) . O f the four classes o f
organotin c o m p o u n d s , diorganotins, R S n X are the largest group o f tin studied for antitumor activity. Dibutyltin dichloride is reported to have in vivo effect
against Ehrlich ascites t u m o r a n d IMC c a r c i n o m a ,
(Crowe, 1987; Crowe et al., 1 9 8 4 ) . Diphenyltin dichloride-3,4,7,8-tetramethyl(-1,10-) phenanthroline is active
against P.388 leukaemia and renal carcinoma (Atassi,
1985).
African t r y p a n o s o m e s are p r o t o z o a n parasites that
cause sleeping sickness in humans and related diseases
in cattle. African trypanosomiasis is considered to b e
o n e o f the most serious diseases affecting both man
and his domestic animals and has b e e n the b a n e
2
against animal production in humid and subhumid tropical Africa.
Chemotherapy is still the main method for controlling
the disease. So far there exists only o n e report o n trypanocidal activity o f an organotin c o m p o u n d (Nok et
al, 1 9 9 2 ) . Herein w e report on the trypanocidal activity of other organotins- dibutyltin dichloride (DBTC) ( C H ) S n C l and diphenyltin dichloride ( D P T C ) (C H ) SnCl .
4
9
6
5
2
2
2
2
2
* Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852, Japan.
** Department of Biochemistry,
*** Department of Chemistry,
**** Department of Veterinary Physiology and Pharmacology,
Ahmadu Bello University, Zaria, Nigeria.
Correspondence: Mohammed Nasir Shuaibu.
Tel.: +81-958-49-7838 - Fax: +81-958-43-2774.
MATERIALS AND METHODS
D
ibutyltin dichloride ( D B T C ) and diphenyltin
dichloride ( D P T C ) were synthesized as described by Van der Kerk & Luijten ( 1 9 5 6 ) . T h e
organotin c o m p o u n d s w e r e dissolved in solutio petit
( 2 6 : 3 3 : 4 2 ) ethanol, glycerol and water respectively as
described b y (William et al., 1 9 7 7 ) .
Six w e e k s old BALB/c m i c e w e r e purchased from the
Department o f Pharmacology and Clinical Pharmacy
Ahmadu Bello University, Zaria and the T. b. brucei (stabilate EATRO 4 1 0 ) was obtained from the Department
o f Veterinary Parasitology and Physiology, Ahmadu
Bello University, Zaria, Nigeria.
T h e mice were intraperitoneally infected with 1 0 trypanosomes. Treatment started six days post-infection and
members of group I and II were intraperitoneally treated
with 10 mg DBTC/kg/day and 15 mg DPTC/kg/day respectively for five consecutive days until the parasi2
43
taemia disappeared. The group III was treated with plac e b o (solutio petit) and served as control. T h e trypanocidal effect of D B T C was assessed b y determining
the level o f parasitaemia every twenty four hours after
treatment. Both dosages w e r e selected from a preliminary work which represented the minimal d o s e that
elicits trypanocidal effect.
T h r e e m i c e e a c h from the D B T C - and DPTC-treated
groups w e r e sacrificed three w e e k s post-treatment.
H o m o g e n a t e s of their liver, spleen, kidney and c e r e brospinal fluid (CSF) w e r e used for subinoculation
e x p e r i m e n t o n healthy mice. About 0.5 ml of b l o o d
was r e c o v e r e d from e a c h o f the treated mice and
used to inoculate three healthy mice each. All tissues
(liver, spleen and kidney) w e r e r e m o v e d under sterile
conditions, washed, weighed and macerated separately
in equal v o l u m e s o f P B S G . Aliquots o f 0.1 ml o f each
o r g a n h o m o g e n a t e in P B S G w a s i n o c u l a t e d i n t o
healthy m i c e . T h e parasitaemia w a s then routinely
c h e c k e d for four w e e k s as described by Nok et al,
1994.
RESULTS
T
h e results are s h o w n in Tables I and II.
Six days post-infection with T. b. brucei,
the
parasitemia in the e x p e r i m e n t a l and control
mice d e v e l o p e d to 10 /ml o f b l o o d . O n the sixth day
of infection the m i c e s h o w e d signs of early trypanosomal infection. O n the c o m m e n c e m e n t of treatment
at 10 mg DBTC/kg and 1 mg DPTC/kg six days postinfection, there w a s an observed gradual d e c r e a s e in
the level o f parasitaemia until the fifth day o f treatment
w h e n n o parasites w e r e o b s e r v e d in the b l o o d .
Table I s h o w s the results o f studies o f relapse three
w e e k s after treatment with 10 mg DBTC/kg and 15 mg
DPTC/kg. 8 3 . 3 % o f the treated mice s h o w e d n o parasitaemia in e a c h batch. T h e control group III s h o w e d
high parasitaemia, and died as a result. Also, of the
apparently cured mice, 23 % in group I and 42 % in
group II w e r e o b s e r v e d to relapse into the infection
with low parasitaemia three w e e k s after treatment
5
Number o f parasitized mice
Group
Liver
Kidney
Spleen
CSF
Blood
DBTC-treated
DPTC-treated
1
1
0
0
0
1
0
0
0
0
The number of DBTC- and DPTC-treated mice killed for subinoculation is three in each treated batch.
Table II. - Parasitized mice following the subinoculation into healthy
mice of tissue homogenate from DBTC- and DPTC-treated mice.
with D B T C and DPTC respectively. This observation,
even with very low parasitaemia, suggests incomplete
recovery, which could b e due to incomplete treatment
regimen. However, it s h o w s the benefit of clearing the
b l o o d o f the parasites as c o m p a r e d to the control
group III.
T a b l e II s h o w s the d e v e l o p m e n t of parasitaemia following the subinoculation of healthy mice with tissue
h o m o g e n a t e s from the D B T C and DPTC-treated mice.
It is o b s e r v e d from the table that n o parasites w e r e
detected in the healthy mice subinoculated with the
h o m o g e n a t e s o f kidney, cerebrospinal fluid (CSF) and
b l o o d o f the D B T C and DPTC-treated m i c e . O n e
healthy m o u s e from e a c h of the three subinoculated
with the liver h o m o g e n a t e o f the DBTC and DPTCtreated mice s h o w e d very low parasitaemia four w e e k s
after subinoculation. Also a m o u s e out of the three
subinoculated with the h o m o g e n a t e of spleen from the
DPTC-treated m o u s e s h o w e d parasitaemia four w e e k s
after subinoculation.
DISCUSSION AND CONCLUSION
F
ollowing the administration of 10 mg DBTC/kg,
six days post-infection for five consecutive days,
trypanosome parasites w e r e eliminated from the
circulation. T h e organotin DPTC at a dose of 15 mg/kg
for five days also cleared the b l o o d o f the parasites
from the onset o f the treatment. T h e logarithm plot o f
parasitaemia against time following treatment, follows
a first order rate kinetics. Extrapolated half life ( t )
values s h o w that the t
o f the parasites in r e s p o n s e
to the effects of D B T C and DPTC are 0.80 and 0.83 day
respectively.
1 / 2
1/2
Studies o f relapses in m i c e t h r e e weeks
post treatment with DBTC and DPTC
Group
I
II
III
No of mice
Aparasitaemic
Cured
12
12
8
83 %
83 %
0 %
67 %
58 %
-
Group I: Mice infected and treated with DBTC 10 mg/kg x 5.
Group II: Mice infected and treated with DPTC 15 mg/kg x 5.
Group III: Infected control.
Table I - Activity of DBTC and DPTC against T. brucei infected mice.
14
T h e trypanocidal activity o f bis(tributyltin o x i d e ) had
previously b e e n reported (Nok et al., 1992). Also it has
b e e n s h o w n that the activity of organotin c o m p o u n d s
d e p e n d s on RxSn moiety ( C r o w e et al., 1984; Barbieri
et al, 1982; Humer et al, 1 9 8 5 ) . In a supportive e x p e riment, subinoculation on the healthy mice with blood,
CSF, and h o m o g e n a t e s o f liver, kidney and spleen o f
the apparently cured m i c e revealed residual parasites
in the liver and the spleen during the aparasitaemic
Note de recherche
Parasite, 2000, 7, 43-45
period. T h e reappearance o f the parasites could b e d u e
to i n c o m p l e t e treatment regimen.
Histopathological
analysis did not s h o w any significant d a m a g e to the
organs o f the D B T C a n d DPTC-treated mice. Moreover
the L D
5 0
of both c o m p o u n d s
is b e t w e e n
75 and
9 0 mg/kg (data not s h o w n ) .
D e v e l o p i n g an effective trypanocidal drug
effective
against all species o f t r y p a n o s o m e s is a difficult task.
Moreso, the s a m e arsenical trypanocides have b e e n
used for the past 5 0 years a n d resistance against these
drugs have recently registered growing f r e q u e n c y in
W e s t e r n and Eastern Africa
( B o u d i c h o n , 1 9 9 8 ) . In an
attempt to exploit the trypanocidal potentials o f organotins, D B T C a n d DPTC w e r e tested a n d found t o b e
trypanocidal against t h e t r y p a n o s o m e s . D B T C a n d
DPTC m a y b e promising c o m p o u n d s in t h e treatment
o f human or animal trypanosomiasis. However, because
o f the i n c o m p l e t e clearance o f t h e T. b. brucei
from
all tissues o f the infected m i c e at the d o s a g e used, a
combination
therapy with o t h e r trypanocidal
com-
p o u n d s could e n h a n c e the efficacy. Synthesis o f organotin c o m p o u n d s with different substituent
groups
other than chloride or m o r e h y d r o p h o b i c c o m p o n e n t s
could b e o f advantage.
notin compound (Tri-n-butyltin oxide) toward T. brucei.
Journal of Clinical Biochemistry and Nutrition, 1992, 13,
81-85.
NOK A.J., IBRAHIM S . , AROWOSAFE S . , LONGDET I., AMBROSE A.,
ONYENEKWE P.C. & WHONG G . C . Z . The trypanocidal effect
of Cannabis sativa constituents in experimental animal trypanosomiasis. Veterinary and Human Toxicology, 1994, 36
(6), 522-524.
PAIN G . & COONEY J . J . Characterization of organotin-resistant
bacteria from Boston harbor sediments. Archives of Environmental Contamination
and Toxicology, 1998, 35 (3),
412-416.
VAN DER KERK G . M . J . & LUIJTEN J . G . A . Investigations on orga-
notin compounds I V . The preparation of a number of
trialkyl and triaryl compounds. Journal of Applied Chemistry, 1956, 6, 49-55.
WILLIAM S., JOSEPH G . V . , VAN SPANJE I.N.E., SNOEK M . , BRANDS R.
& HOOYKAAS H . Toxicity of organotin compounds. II. Comparative in vivo and in vitro studies with various organotin
and organolead compounds in different animal species
with special emphasis on lymphocyte cytoxicity. Toxicology and Applied Pharmacology,
1977, 42, 197-212.
Reçu le 8 octobre 1999
Accepté le 13 décembre 1999
REFERENCES
ATASSI G. Antitumor and toxic effects of Silicon,Germanium,
Lead and Tin compounds. Rev. Si, Ge, Sn, Pb compounds,
1985, 8, 219-235.
BARBIERI R.P., RUISI L.G. & LA GUIDICE M.T. The antitumor acti-
vity of diorganotin(iv) complexes with adenine and glycylglycine. Inorganic Chemica Acta, 1982, 66, 39-40.
BRIDDLE B.N. & GRAY J.S. Structural effect on the antitumor
activity of a series of di (4-substituted) phenyltin dichloride complex with nitrogen-donor ligands. Applied Organometallic Chemistry, 1989, 3, 537-543.
BOUDICHON A. J . Report on the use of the trypanocidal drug
"TRYPAN". Journal of Protozoological
Research, 1998, 8,
258-262.
CROWE J.A. The chemotherapeutic properties of tin compounds. Correlates in pharmacostructures ITRI publication,
1987, No. 676, pp. 255-275
CROWE A. J . , SMITH P.J. & ATASSI G. Investigation into the anti-
tumor activity of organotin compounds 2. Diorganotin
dichloride and dipseudohalide complexes. Inorganic
Chemica Acta, 1984, 93, 179-184.
HUMER F . , ROGE G.L., ATASSI G., SPREAFICO F . , FILIPPESCHI S.,
BARBIERI R., SILVESTRI A., RIVAROLA E., RUISI G., DIBIANCA F .
& ALANZO G. Studies on the antitumor activity of di- and
tri-organotin(iv) complexes of amino acids and related
compounds, of 2-mercaptoethane sulphonate and o f
purine-6-thiol. Journal of Chemical Society Dalton Transactions, 1985, 523-527.
NOK A.J., KING A.N.E., ADAUDI A., ACHOBA I.I., GIMBA C.E.,
MUSA O.S. & KAGBU J.A. Trypanocidal activity of an orga-
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