R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d ic in a T r o p ic a l 2 5 ( 4 ) : 2 4 7 - 2 5 0 , o u t- d e z , 1 9 9 2
G LUCANTIM E RESISTANT L E IS H M A N IA PROMASTIGOTES
ARE SENSITIVE TO PENTOSTAM
Elizabeth Spangler Andrade M oreira, Juliana Becattini Guerra and M aria de Lourdes Petrillo-Peixoto
G r o w th in h ib itio n in vitro te s ts w e r e u s e d to s tu d y th e s u s c e p tib ility to p e n to s ta m o f d if f e r e n t L eish m a n ia s tr a in s in v o lv e d in c u ta n e o u s a n d m u c o c u ta n e o s le is h m a n ia s is - o n e g lu c a n tim e s e n s itiv e s tr a in , th r e e n a tu r a lly g lu c a n tim e r e s is ta n t s tr a in s a n d o n e g lu c a n tim e r e s is t a n t lin e d e v e l o p e d b y in vitro d r u g e x p o s u r e . C o n tr a s tin g w ith th e h ig h d e g r e e , o f g lu c a n tim e r e s is ta n c e , a l l s tr a in s w e r e s e n s itiv e to p e n to s ta m . T h e s e d if f e r e n c e s s u g g e s t th a t th e r e is s o m e r e la tio n s h ip b e tw e e n c h e m ic a l s tr u c tu r e a n d in vitro a c tiv ity f o r th e s e a n tim o n ia l c o m p o u n d s . T h e se d a t a ju s ti f y a c lin ic a l r e - e v a lu a tio n to c o m p a r e th e r a p e u tic e ff ic a c y o f g lu c a n tim e a n d p e n to s ta m in th e tr e a tm e n t o f le is h m a n ia s is .
K e y - w o r d s : L eish m a n ia P e n ta v a le n t E x p e r im e n ta l c h e m o th e r a p y .
Antimony-unresponsiveness in visceral and mucocutaneous leishmaniasis are increasing in severity and prevalence worldwide5-6 14 1719. It is not known if antimonial drug insensitivity is attributable to inherent or developed resistance of the parasite, in addition to a variety of host factors that also contribute to chemotherapeutic relapse.
The pentavalent antimony (Sb) complexed to a carbohydrate in the form o f sodium stibogluconate (p e n to sta m ) o r m eg lu m in e an tim o n ia te (g lu c a n tim e ) is th e on ly an tile ish m a n ia l chemotherapeutic agent with clearly favorable therapeutic index. Pentostam, produced in the United Kingdom, is used there, in USA and in most African and Midle-East countries; glucantime, produced in France and in Brazil, is used in french speaking countries and in Latin America. Different dosage schedules have been recommended6 81314, considering the variation o f treatment among the various forms o f human leishmaniasis and the importance o f the relapse problem. Generally, all
D epartam ento de M icrobiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG.
Research supported by PRPq/UFM G.
This work was perform ed in partial fulfillment o f the requirem ents for the P h.D . (E .S .A .M .) at Instituto de Microbiologia da Universidade Federal do Rio de Janeiro. Address to: Dra. Elizabeth S.A. Moreira. Laboratório de Biologia de Microbiologia/ICB/UFM G, CP: 2486, 31271- 970 Belo Horizonte, MG
Recebido para publicação em 28/05/92.
a n tim o n ia l c o m p o u n d s . G r o w th in h ib itio n .
drug doses are expressed in milligrams of Sb per kilogram per day and the therapeutic schemes are standardized irrespectively o f the preparation312. On theoretical basis, it is assumed that both drugs have equivalent therapeutic indexes, i.e., th eS binthetw o formulations are equally rem ovable from the respective carbohydrates. There are no reports of c lin ic a l stu d ies co m p arin g th e th e ra p e u tic effectiveness o f glucantime and pentostam applied in the same dose3 12.
Nevertheless, differences in the susceptibility between both drugs are observed, when glucantime is included in in v itr o comparative tests for Sb activity: usually pentostam is more active than glucantim e1 11 18. These studies indicate that L e is h m a n ia strains may have different susceptibility profiles for each drug, although no rationale for these variations is presented.
A re-evaluation o f this problem is necessary since pentavalent antimonials remain the drugs of choice for leishmaniasis. Here, w e report that promastigotes o f glucantime resistant L e is h m a n ia strains, involved in cutaneous and mucocutaneous leishmaniasis, are sensitive to pentostam, when tested under identical conditions.
MATERIAL AND METHODS
The L e is h m a n ia ( V .) b r a z i l i e n s i s , strains MHOM/BR/75/M29Q3, MHOM/BR/??/LTB259,
M o r e i r a E S A , G u e r r a J B , P e tr i ll o - P e ix o t o M L . G lu c a n tim e r e s i s t a n tLeishmania p r o m a s t i g o t e s a r e s e n s i t i v e to p e n t o s t a m . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c in a T r o p ic a l 2 5 : 2 4 7 - 2 5 0 , o u t- d e z , 1 9 9 2 .
ISQN/BR/85/M 9947 and the L e is h m a n ia (L .) a m a z o n e n s is IFLA/BR/86/M 10995 were used in these experiments. With the exception of theM2903, all strains were previously shown to be resistant to glucantime16. A glucantime-resistant line (R2-1) derived from the sensitive/,. (V .) g u y a n e n s is IUMB/ BR/85/M9945 strain, obtained by in v itr o drug exposure to high glucantime concentration15, was also evaluated. The strains have been cryopreserved as well as maintained by serial passage in logarithmic phase at 25 °C in a liquid complex medium supplement with 40% o f whole rabbit blood10.
Glucantime (meglumine antimoniate, 85mg Sb m l'1, batch 020, Rhodia SA, Brazil) and pentostam (sodium stibogluconate, lOOmg Sb m l'1, bacth C54262, WellcomeFoundation Ltd, England) were stored in the dark, at room temperature. Fresh drug samples w ere used in each experiment. The concentration o f these drugs is given as mg of pentavalent antimony according to the manufactures ’ information.
The sensitivities o f different L e is h m a n ia strains to both antimonial drugs, based on promastigotes growth inhibition, were determined as previously described16. Briefly, 25x10s parasites in 5.0ml of fresh m edium w ere incubated with different c o n c e n tra tio n s o f th e drug (p en to stam or glucantime). Cultures of each L e is h m a n ia strains lacking drug were maintained in parallel as control. The number o f parasites was determined by counting in a Coulter Counter (Model D2) and all results w ere expressed as the mean o f at least two experiments in duplicate. The effect of drug on cell yield was expressed as the percentage o f cell number related to control cultures at late log phase.
RESULTS AND DISCUSSION
In previous studies16 w e demonstrated that the M 9947, M 10995 and LTB259 L e is h m a n ia strains are naturally resistant to glucantime: no significative growth effect could be observed in presence of glucantime concentration below 2.0mg Sb/ml - a drug concentration which showed high growth inhibition o f sensitive cells. The response of different strains to pentostam and glucantime in the present comparative study is summarized in Table 1. The data show that these strains are sensitive to pentostam. Even in the case of M 10995 strain, the
T a b le 1 - S u s c e p tib ility o f d if f e r e n t Leishmania.v/ra/'«.? to a n tim o n ia l d r u g s .
D ru g (m g S b /m l)
P ro m a stig o te su rv iv a l*
M 2 9 0 3 M 9 9 4 7 L T B 2 5 9 R 2-1 M 1 0 9 9 5
P entostam
0 .2 5 4 12 15 9 84 0 .5 0 2 6 13 7 • 5 5 1 .0 0 2 7 12 6 3 7 G lu ca ntim e
2 .0 0 5 7 8 63 7 3 9 8
* P ercen t o f grow th at drug co n cen tra tio n (m g S b /m l) related to th e con trol c e ll n um ber at la te lo g p h a se.
dose of pentostam which gave rise to about 50 % in growth inhibition was one fourth o f that o f glucantime used without response. Furthermore, a cell line derived from L . (V .) g u y a n e n s is , selected in laboratory for glucantime resistance, showed very little response to 2mg Sb/ml in the form of glucantime, whereas a high growth inhibition was observed in the presence of pentostam, even at lowest concentration tested (0.25mg Sb/ml).
These data are simultaneously disturbing and exciting. They may suggest that there is indeed some relation between chemical structure and in v itr o activity for these antimonial compounds.
Since glucantime and pentostam chemical structures and their biochemical mechanisms o f antileishmanial activity are unknown it can be proposed that the two compounds may have different mechanisms of action. On the other hand, the influence o f carb o h y d rate m oiety on antim ony activ ity demonstrated when the leishmanicidal effects of antimony coupled to yeast mannan were compared to those obtained with glucantime2 may suggest that a differential kinetics of drug accumulation and Sb release could account for the differences observed. U n fo rtu n ately , rad io lab eled p en to stam and glucantime necessary to evaluate the capacity of cells to transport the drugs is commercially unavailable.
Another possibly explanation should consider that L e is h m a n ia , as other parasites, has the potential to respond to drug pressure in multiple ways, resulting in drug resistance4. So, the different drug susceptibility profiles observed between pentostam and glucantime may involve different mechanisms of resistance. Drug resistance in L e is h m a n ia has
M o r e i r a E S A , G u e r r a J B , P e tr i ll o - P e ix o t o M L . G lu c a n tim e r e s i s t a n t L e ish m a n ia p r o m a s t i g o t e s a r e s e n s i t i v e to p e n t o s t a m . R e v i s t a d a S o c ie d a d e B r a s i le i r a d e M e d ic in a T r o p ic a l 2 5 : 2 4 7 - 2 5 0 , o u t- d e z , 1 9 9 2 .
been studied with some drugs4 but only few studies have been done with antimonial compounds7 1118 and very little is known about the mechanisms of antimonial resistance.
Although drug sensitivity data obtained with promastigotes in v itr o cannot be directly extrapolated to the situation in v itr o , these results justify a clinical evaluation in cutaneous and mucocutaneous leishmaniasis, once very seldom pentostam is considered as an alternative after glucantime failure9.
RESUMO
D if e r e n te s a m o s tr a s d e L eish m ania f o r a m a n a lis a d a s q u a n to â s u s c e p tib i li d a d e in vitro a o p e n to s ta m - u m a c e p a d e L . (V ) b ra zilien sis c o n s id e r a d a s e n s ív e l a o g lu c a n tim e , tr ê s c e p a s (d u a s L . (V ) b ra zilien sis e u m a L. (L) a m a z o n e n sis) c o n s i d e r a d a s n a tu r a lm e n te r e s is te n te s a o g l u c a n t i m e , u m a lin h a g e m r e s i s t e n t e (L . (V ) g u y a n e n sis) s e l e c io n a d a in vitro p e l a e x p o s iç ã o e m a lta c o n c e n tr a ç ã o d e d r o g a . A e le v a d a s e n s ib ilid a d e d e s ta s a m o tr a s e m c o n tr a p o s iç ã o à r e s is tê n c ia o b s e r v a d a p a r a o g lu c a n tim e s u g e r e e x is tir r e l a ç ã o e n tr e a e s tr u tu r a q u ím ic a e a a ti v id a d e d e s t e s c o m p o s to s . E s te s d a d o s in d ic a m a n e c e s s id a d e d e i m a a v a lia ç ã o c o m p a r a tiv a d e a ti v id a d e c lín ic a d o p e n to s ta m e d o g lu c a n tim e n o tr a ta m e n to d a le is h m a n io s e .
P a l a v r a s - c h a v e s : L e i s h m a n i a . c o m p o s t o s a n tim o n ia is p e n ta v a le n te s . I n ib iç ã o d e c r e s c im e n to . I n f e c ç ã o e x p e r im e n ta l.
ACKNOWLEDGMENTS
We thank Drs P. D. Marsden, Universidade de Brasília, andR. Lainson, Instituto Evandro Chagas, Belém, for providing strains o f L e is h m a n ia , and to A. L. Miranda Vilella for pro vinding the glucantime resistant cell line. The technical support of M.A. Santos is gratefully acknowledged.
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