The activity of tricyclic antidepressant drugs against Trypanosoma cruzi

EXPERIMENTAL

PARASITOLOGY

The Activity

68, 230-234 (1989)

of Tricyclic

Antidepressant

Drugs against

Trypanosoma cruzi PATRICIAS.DOYLEANDEUGENE Laboratory

of Parasitic

Diseases, National Institute of Health, Bethesda,

of Allergy Maryland

C. WEINBACH' and Infectious 20892, U.S.A.

Diseases,

National

Institutes

DOYLE, P. S., AND WEINBACH, E. C. 1989. The activity of tricyclic antidepressant drugs against Trypanosma cruzi. Experimental Parasitology 68, 23&234. Imipramine and related derivatives were tested as possible chemotherapeutic agents against Trypanosoma cruzi parasites in vitro. The I&, values and the lethal concentrations for two cloned stocks of the parasite were determined. 2-Nitrodesmethylimipramine was the most effective compound tested (IC, = 4-7 p&f). Parasites that were able to grow and to complete the intracellular cycle in mammalian cells in the presence of the drug could be selected. Differences in susceptibility to some imipramine analogs between T. cruzi-cloned stocks were found. The study also shows that modification of the imipramine molecule by electron-withdrawing groups greatly enhances its biological activity. o 1989 Academic press, IIIC. INDEX DESCRIPTORS AND ABBREVIATIONS: Trypanosoma cruzi; Kinetoplastida; Protozoa, parasitic; Chagas’ disease; Tricyclic antidepressant drugs; Imipramine and its analogs; Bovine embryo skin and muscle cell (BESM); Fetal calf serum (FCS); Liver infusion tryptose (LIT) medium.

Chagas’ disease caused by the protozoan parasite Trypanosoma cruzi, is a major health problem in South and Central America. No entirely suitable chemotherapeutic agents are available for either the treatment or the prevention of the disease. The action of a group of amphiphilic cationic compounds, the antidepressant imipramine and its metabolites, has been extensively studied in protozoa which infect man. They have been found to inhibit the in vitro growth of Giardia lamblia (Weinbach et al. 1985) Naegleria, and Acanthamoeba (Schuster and Mandel 1984), to be lethal for Leishmania sp. promastigotes and intracellular amastigotes (Pearson et al. 1982; Zilberstein and Dwyer 1984), and to lyse the trypomastigote and epimastigote stages of T. cruzi (Hammond et al. 1984, 1985; Barioglio et al. 1987). These findings suggest that tricyclic drugs may be useful in the ’ To whom correspondence should be addressed at NIH, Building 4, Room 126, 9000 Rockville Pike, Bethesda, MD 20892.

prevention and/or treatment of an infection by some parasitic protozoa. In order to further test this concept, we studied the effect of imipramine and three of its analogs on the in vitro growth of T. cruzi. MATERIALSAND METHODS Trypanosoma cruzi and vertebrate cell cultures. The epimastigote stage of T. cruzi-cloned stocks Sylvio Xl014 (SXlO/4) (Postan et al. 1983) and Miranda/88 (M/88) (Engel et al. 1982) was grown in LIT medium (Bone and Steinert 1956) containing 20 pg/ml hemin, 100 U/ml penicillin, 100 &ml streptomycin, and 10% heat-inactivated FCS at 26 C.BESM (Flow Laboratories, McLean, VA, U.S.A.) were maintained in RPM1 1640 medium supplemented with 10% heatinactivated FCS in a humified atmosphere of 95% aid% CO, at 37 C. Drug testing. Imipramine (Sigma Chemical Co., St. Louis, MO, U.S.A.), 3-chlorimipramine (courtesy of Ciba-Geigy Corp., Summit, NJ, U.S.A.), 2nitroimipramine, and 2-nitrodesmethylimipramine (generous gifts from Dr. K. Rice, Laboratory of Chemistry, NIADDK, NIH, Bethesda, MD, U.S.A.) were dissolved in dimethyl sulfoxide at a concentration of 60 nG4. Exponential growth phase epimastigotes were harvested and resuspended at a concentration of lo6 parasites/ml in LIT medium (controls) or in LIT medium with increasing concentrations of the compound 230

0014-4894189 $3.00 Copyright

0 1989 by Academic Press, Inc. any form reserved.

AUr&its of reproductionin

T. cruzi: TRICYCLIC ANTIDEPRESSANT DRUGS

231

imipramine under identical conditions; the I&, was 12 $ and 20 pM was lethal for the parasites. 2-Nitroimipramine in concentrations >50 pM was tidal for SX10/4 and at a concentration of 50 pJ4 for M/88 epimastigotes. The I&, values were 25 and 15 @!4, respectively. 2-Nitrodesmethylimipramine was the most toxic compound tested. The ICsO values were 7 and 4 kit4 for clones SX10/4 and M/88, respectively. No growth of SX10/4 epimastigotes occurred at a 10 PM concentration of the drug for the first 7-11 days. Parasite populations (ElO) then resumed slow growth with a doubling time of 64.4 hr (Table II, Fig. 1). A 20 pA4 concentration was lethal for the SX10/4 cells. All compounds induced the appearance of concentration-dependent morphological alterations in the epimastigotes. Cells rounded up, formed clumps, and either lost or exhibited reduced motility (data not shown). Concentrations of DMSO equivalent to those required to solubilize the drugs had no effect upon population growth. Clone SX10/4 epimastigotes gradually adapted to grow over a period of 6 months RESULTS in the presence of increasing concentraThe effects of tricyclic compounds on the tions of 2-nitrodesmethylimipramine. Parasite morphology appeared normal once adgrowth of Trypanosoma cruzi epimastidoubling gotes in LIT medium are summarized in Ta- aptation occurred. Population ble I. Imipramine at concentrations up to 50 times and the percentage of growth inhibitGV had no effect no SX10/4 and Ml88 epi- tion of parasite populations serially subculmastigote growth. In contrast, 3- tured in the presence of 2-nitrodesmethylimipramine and controls are presented in chlorimipramine markedly inhibited growth of SX10/4 epimastigotes: I&a = 16 @t4. A Table II and Fig. 1. The same protocol was 20 p.M concentration initially inhibited used to obtain resistant lines of clones M/88 growth completely. Between Days 5-9, and other T. cruzi clones at concentrations transferred from however, parasite growth resumed with an lethal for epimastigotes estimated population doubling time of 107.6 LIT medium (data not shown). The survival hr. Although no growth was observed at of epimastigotes at drug concentrations beconcentrations 330 pfU, parasites trans- tween 30 and 45 p.M was influenced by parferred to drug-free LIT medium following a asite concentrations. Cultures initiated with lo7 cells/ml could be serially maintained for 20-day exposure to 30 PM 3-chlorimipramine resumed growth. A 50 FM con- a period of 3 months. However, attempts to establish cultures at lower parasite concencentration was lethal. Clone M/88 epimastitrations were not successful. SX10/4 epigotes were more sensitive to 3-chlor-

tested. Epimastigote growth studies were performed in duplicate; five different drug concentrations were tested in each independent study. Epimastigote growth was estimated with a Coulter Counter (Model ZB,, Coulter Electronics, Hialeah, FL, U.S.A.) for 4 to 30 days at preselected times dependent upon the growth rate of the treated cultures. Growth kinetic data were analyzed with an IBM FC computer using software developed specifically for this purpose. Qualitative studies of all cultures were performed daily by phase contrast light microscopy. The percentage of growth inhibition was calculated as (1 - growth rate of the experimental culture/growth rate of the control culture) x 100. The drug concentration required to inhibit growth by 50% (I&,) was determined by linear regression analyses. The drug concentration required to kill 100% of the parasites within 48 hr (lethal concentration) was assessed by phase contrast microscopy. Both the SX10/4 and M/88 parasite stocks were subjected to 5 pM concentration increments in a stepwise procedure from 10 to 45 pit4 of 2-nitrodesmethylimipramine over a period of 6 months in an attempt to develop drug-resistant parasite stocks. BESM cell cultures were infected in the presence of 2-nitrodesmethylimipramine with stationary-phase SX10/4 organisms from drug-treated cultures in LIT medium. Trypomastigotes released from BESM cells in drug-treated cultures were used to serially passage the parasite stock. The toxicity of 2-nitrodesmethylimipramine and 3-chlorimipramine for BESM cells was estimated qualitatively by phase contrast light microscopy.

232

DOYLE

AND

TABLE

WEINBACH

I

Effect of Tricyclic Compounds on the Growth of Ttypanosoma cruzi Epimastigotes in Vitro

Drug

Clone

Lethal Concentration t/M

>50 >50

>50 >50

SXlOl4 Ml88

16il 12 io.3

50 20

sx1014 Ml88

25i1.6 15zto.4

>50 50

sx1014 Ml88

7*0.1 4kO.2

20 7.5

lmipramine

3-Chlorimipramine

I&,, (/AM) f S.E.M.

CH&H,CH,N(CH& 2-Nitroimipramine

NO2

3

CH2CH2CH2NCHJ 2-Niirokmethylimipramine

mastigotes serially maintained in 20 t&f 2nitrodesmethylimiprine could be serially cultured in 30 pA4 chlorimipramine. Metacyclic trypomastigotes present in stationary-phase axenic cultures grown in the presence of 20 kA4 2nitrodesmethyl imipramine infected BESM cells, completed the intracellular cycle, and could be serially maintained either in the presence of

20 ~&II 2nitrodesmethylimipramine or 3chlorimipramine. Higher concentrations of the drugs were toxic for the cultured mammalian cells. DISCUSSION

Tricyclic antidepressant drugs are highly lipophilic compounds known to induce lipidosis, probably as a consequence of inter-

T. cruzi: TRICYCLIC

ANTIDEPRESSANT

TABLE II Effect of 2-Nitrodesmethylimipramine on the Growth Rates of Four Trypanosoma cruzi SXlOl4 Stocks Parasite stock

Doubling time SEM (hd

Control LlV L206 EIOC

22.8 33.4 55.1 64.4

Growth inhibition (%I

Growth rate 0.82 0.58 0.29 0.26

+ 0.6 + 2.5 + 4.6 rfr 5.7

0 29 65 68

o SX1014 epimastigotes maintained in LIT medium with 10 @V 2-nitrodesmethylimipramine for 340 generations. b SX1014 epimastigotes maintained in LIT medium with 20 pM 2-nitrodesmethylimipramine for 210 generations. ’ SX1014 epimastigotes transferred from LIT medium to the same medium with a 10 pA4 concentration of the drug.

action with phospholipids (Lullman et al. 1978), destabilizing membrane structures, and accumulating in lysosomes (Fauster et al. 1983). Our data show that imipramine derivatives effectively suppressed the growth of Trypanosoma cruzi epimastigotes in vitro, a method useful for a primary screening of drugs active against this parasite (Schlemper et al. 1977). Imipramine substituted at either the 2- or 3- position results in a more active compound than the parent drug (cf. Table I). 3Chlorimipramine was tidal for both T. cruzi clones at lower concentrations than those reported for the Sonya strain (Hammond et al. 1984), l

Control

LlO

l

5

10

l

L20

a

El0

15

DAYS

FIG. 1. Effect of 2-nitrodesmethylimipramine on the growth rates of four Trypanosoma cruzi SX10/4 stocks. Conditions as described in Table II.

DRUGS

233

probably due to the difference in the methodology employed or to differences in the susceptibility of the parasite stocks used. Such differences were observed in the course of this study. The novel compound 2nitrodesmethylimipramine was the most effective compound tested. However, as shown in Fig. 1, parasites resistant to initially lethal concentrations of the drug could be selected. Such parasites were able to complete the intracellular cycle in the presence of the drug or one of its analogs. Survival at higher drug concentrations (>20 pA4) depended on parasite density. Hammond er al. (1985) demonstrated a similar effect for thioproperazine, another amphiphilc cationic drug. It is a well-documented phenomenon that many drugs are more active on exponentially growing eukaryotic cells (i.e., less dense) than on stationary cells (see review by Drewinko and Barlogie 1982). The growth-suppressing activity and lethality of tricyclic antidepressant drugs on T. cruzi and other parasitic protozoa are of particular interest because these drugs have been extensively used for treatment of psychiatric disorders without serious adverse side effects (Linnoila et al. 1982). Their use to prevent Chagas’ disease by blood transfusion and as chemotherapeutic agents has been proposed (Hammond et al. 1985; Barioglio et al. 1987). Our results suggest that 2-nitrodesmethylimipramine might prove useful in this regard in the prevention of infection with T. cruzi by blood transfusion. Development of resistance, however, must be considered in further evaluation of tricyclic compounds as chemotherapeutic agents in Chagas’ disease. Although the mechanism of the tidal action of tricyclic compounds is not known, interaction with biomembranes is a likely key factor in the activity of these lipophilic compounds against Trypanosoma cruzi as suggested by the marked alteration in parasite morphology. The present study also extends our earlier observations (Weinbach

DOYLEANDWEINBACH

234

et al. 1985, 1986) that chemical modification of the parent compound by the addition of electron-withdrawing moieties (such as Cl-, NO-*) intensifies its biological activity. Further chemical modifications of the imipramine molecule may produce chemotherapeutic compounds for the treatment of this major disease. ACKNOWLEDGMENTS The authors express their appreciation to Drs. J. A. Dvorak and J. C. Engel for helpful discussions.

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Received 13 June 1988; accepted with revision 27 October 1988