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Acta Tropica, 48(1991)223-232 223 Elsevier ACTROP 00118 Evaluation of DL- -difluoromethylornithine against susceptible and drug-resistant Trypanosoma brucei brucei E. Zweygarth 1'2 and R. Kaminsky 3 1Kenya Trypanosomiasis Research Institute ( KETRI) , Kikuyu, Kenya; 21nstitutfiir Parasitologie und Tropenveteriniirmedizin, Freie Universitdt Berlin, Berlin, ER.G., and 3International Laboratoryfor Research on Animal Diseases (ILRAD), Nairobi, Kenya (Received 23 March 1990; accepted 28 May 1990) The antitrypanosomal activity of the ornithine decarboxylase inhibitor DL-c~-difluoromethylornithine (DFMO, eflornithine) was tested in ten stocks and one clone of the hemoflagellate Trypanosoma brucei brucei in an in vitro system. They showed varying levels of susceptibility to DFMO, their IC5o (the concentration which inhibited growth by 50%) values ranging from 81-691 laM. Differences in DFMO susceptibility were also demonstrated in mice. Combinations of melarsonyl potassium (mel W; trimelarsan) and DFMO showed an additive effect in vitro in a mel W-susceptible and a met W-resistant stock, but an antagonistic effect in a mel W- and DFMO-susceptible clone, Combinations of suramin and DFMO showed an antagonistic effect in vitro in a suramin-susceptible clone, but a potentiation in a suramin- resistant stock. Key words: Trypansoma brucei brucei, In vitro cultivation; Drug sensitivity; Drug synergy; DL-~-Difluoromethylornithine; DFMO; Eflornithine; Mel W; Suramin Introduction Chemoprophylaxis and chemotherapy are the methods of control of African trypano- somiasis. The appearance of drug-resistant trypanosomes, however, has reduced the value of the existing drugs and thus new compounds are urgently needed. One of the more promising compounds is oL-~-difluoromethylornithine (DFMO; eflornithine; Ornidyl R) a selective, enzyme-activated irreversible inhibitor of ornithine decarboxyl- ase, a key enzyme for the synthesis of polyamines (Metcalf et al. 1978). It is effective against Trypanosoma brucei brucei (Bacchi et al., 1980), T.b. gambiense (McCann et al., 1981a), T.b. rhodesiense (McCann et al., 1981b) and T. congolense (Karbe et al., 1982) infections in mice. Difluoromethylornithine acts synergistically with a variety of commercial trypa- nocides against early stages of murine T.b. brucei infections (McCann et al., 1983). Combinations of DFMO and suramin (Clarkson et al., 1984), DFMO and melarso- prol (Jennings, 1988), and DFMO and diminazene aceturate (our unpublished results) are effective even in cases with central nervous system (CNS) involvement. Correspondence address." Dr. E. Zweygarth, P.O. Box 29 231, Nairobi, Kenya. 0001-706X/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

Evaluation of dl-α-difluoromethylornithine against susceptible and drug-resistant Trypanosoma brucei brucei

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Page 1: Evaluation of dl-α-difluoromethylornithine against susceptible and drug-resistant Trypanosoma brucei brucei

Acta Tropica, 48(1991)223-232 223 Elsevier

ACTROP 00118

Evaluation of DL- -difluoromethylornithine against susceptible and drug-resistant Trypanosoma

brucei brucei

E. Zweygarth 1'2 and R. Kaminsky 3

1Kenya Trypanosomiasis Research Institute ( KETRI) , Kikuyu, Kenya; 21nstitut fiir Parasitologie und Tropenveteriniirmedizin, Freie Universitdt Berlin, Berlin, ER.G., and 3International Laboratory for

Research on Animal Diseases (ILRAD), Nairobi, Kenya

(Received 23 March 1990; accepted 28 May 1990)

The antitrypanosomal activity of the ornithine decarboxylase inhibitor DL-c~-difluoromethylornithine (DFMO, eflornithine) was tested in ten stocks and one clone of the hemoflagellate Trypanosoma brucei brucei in an in vitro system. They showed varying levels of susceptibility to DFMO, their IC5o (the concentration which inhibited growth by 50%) values ranging from 81-691 laM. Differences in DFMO susceptibility were also demonstrated in mice. Combinations of melarsonyl potassium (mel W; trimelarsan) and DFMO showed an additive effect in vitro in a mel W-susceptible and a met W-resistant stock, but an antagonistic effect in a mel W- and DFMO-susceptible clone, Combinations of suramin and DFMO showed an antagonistic effect in vitro in a suramin-susceptible clone, but a potentiation in a suramin- resistant stock.

Key words: Trypansoma brucei brucei, In vitro cultivation; Drug sensitivity; Drug synergy; DL-~-Difluoromethylornithine; DFMO; Eflornithine; Mel W; Suramin

Introduction

C h e m o p r o p h y l a x i s and c h e m o t h e r a p y are the me thods o f con t ro l o f Af r ican t rypano- somiasis . The appea rance o f d rug- res i s tan t t rypanosomes , however , has reduced the value o f the exist ing drugs and thus new c o m p o u n d s are urgent ly needed. One o f the more p romis ing c o m p o u n d s is oL-~-d i f luoromethy lo rn i th ine ( D F M O ; eflornithine; Orn idyl R) a selective, enzyme-ac t iva ted i r revers ible inh ib i to r o f orn i th ine deca rboxy l - ase, a key enzyme for the synthesis o f po lyamines (Metca l f et al. 1978). I t is effective agains t Trypanosoma brucei brucei (Bacchi et al., 1980), T.b. gambiense ( M c C a n n et al., 1981a), T.b. rhodesiense ( M c C a n n et al., 1981b) and T. congolense ( K a r b e et al., 1982) infect ions in mice.

D i f luo romethy lo rn i th ine acts synergis t ical ly with a var ie ty o f commerc ia l t rypa- nocides agains t ear ly stages o f mur ine T.b. brucei infect ions ( M c C a n n et al., 1983). C o m b i n a t i o n s o f D F M O and suramin (C la rkson et al., 1984), D F M O and melarso- prol (Jennings, 1988), and D F M O and d iminazene ace tura te (our unpubl i shed results) are effective even in cases with central nervous system (CNS) involvement .

Correspondence address." Dr. E. Zweygarth, P.O. Box 29 231, Nairobi, Kenya.

0001-706X/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

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DF MO has been extensively used against the Gambian form of human sleeping sickness with promising results. D F M O was effective against the early stages of the disease (Van Nieuwenhowe et al., 1985; Taelman et al., 1987), and against the late stages (Van Nieuwenhouwe et al., 1985; Doua et al., 1987; Pepin et al., 1987; Taelman et al., 1987). Moreover, it was effective against arsenical-refractory cases of T.b. gambiense infections in humans (Van Nieuwenhowe et al., 1985; Doua et al., 1987; Pepin et al., 1987; Taelman et al., 1987).

The present paper deals with the efficacy of DFMO, alone or in combination with melarsonyl potassium (mel W; trimelarsan) or suramin, against a variety of T.b. brucei stocks with different susceptibility patterns to commercial trypanocides in mice under defined in vitro conditions.

Materials and Methods

Trypanosome stocks

Ten stocks and one clone of T. brucei brucei were used. Their designation, place and date of primary isolation, and the animal species from which they were isolated is summarized in Table 1. The stocks, except CP 2711, were screened in mice for their susceptibility to diminazene aceturate (15 and 30 mg/kg), isometamidium chloride (3 mg/kg) and suramin (20 and 40 mg/kg) (Table 2). Clone ILTat 1.4 is derived from stock EATRO 795, which was isolated in 1964 from a bovine in Uhembo, Kenya. This clone was susceptible in mice to diminazene aceturate, isometamidium chloride and suramin at the above concentrations.

Test for human serum resistance

All stocks were tested for their resistance to human serum, according to the method of Jenni and Brun (1982) as modified by Zweygarth and R6ttcher (1988). A further modification of the technique was employed by using feeder layer-free culture condi- tions. Briefly, trypanosomes were cultivated in 24-well culture plates. The horse serum in the medium (as described below) was replaced by 15% heat-inactivated

TABLE 1

Origin of the trypanosome stocks

Designation Place/year of isolation Animal species

CP 161 Ngurunit, Kenya/unknown Camelid CP 271 Matuga, Kenya/1980 Caprine CP 547 Jilib, Somalia/1985 Bovine CP 1154 Korbesa, Kenya/1982 Camelid CP 1184 Rumuruti, Kenya/1981 Camelid CP 1284 Taru, Kenya/1984 Equine CP 2107 Mogadishu, Somalia/1985 Bovine CP 2 137 Nairobi, Kenya/1986 Canine CP 2469 Hakaka, Somalia/1985 Bovine CP 2711 Lodwar, Kenya/1987 Camelid

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TABLE 2

Susceptibility of Trypanosorna brucei brucei stocks to diminazene aceturate, suramin in mice

225

isometamidium chloride and

Trypanosome Diminazene Isometamidium Suramin stock aceturate chloride

(3mg/kg) 20mg/kg 40mg/kg 15 mg/kg 30 mg/kg

CP 161 R (1/8) ~ R S S CP 271 S S S S S CP 547 R R R S S CP 1154 S S S R R CP 1184 S S S S S CP 1284 S S (5/8) S S CP 2107 R R S R R CP 2137 S S S S S CP 2469 R R R S S

R = None of the mice were cured with the doses indicated; S = All mice were cured with the doses indicated. ~Figures in brackets indicate number of cured/number of treated mice. nd = experiment not done.

(56°C, 30 min) human serum. To exclude the possibility that human serum was lacking in essential growth factors for the trypanosomes, control cultures were included with 15% human serum as well as 10% horse serum, or with 10 and 20% (v/v) horse serum alone in the medium. All tests were carried out in triplicate.

Culture medium

Bloodstream form trypanosomes were cultivated in modified Eagle's MEM medium (Gibco, Cat. No 041-02360 M, Paisley, U.K.) with 20% heat-inactivated (56°C, 30 min) horse serum, 1% MEM non-essential amino acids (Gibco, Cat. No 043-01140 H), containing 2 mM L-glutamine, 100 IU/ml penicillin, I00 ~tg/ml streptomycin, and 0.1 mM hypoxanthine. 2-merceptoethanol was added at a final concentration of 0.2 mM according to Baltz et al. (1985).

Initiation of trypanosome cultures

Cultures were initiated by the method of Zweygarth et al. (I 989). Briefly, blood was obtained from infected mice by disinfecting the tail with surgical spirit and cutting the tip. Drops of blood were aspirated with a 5 ~tl Eppendorf pipette and then transferred each into a well of a culture plate (24-wells plate, COSTAR, Cambridge, MA, U.S.A.). Some wells contained a fetal bovine thymus fibroblastoid feeder layer cell line and others were cell-free. All wells contained 1 ml of medium. The blood was deposited in the corner at the bottom of the wells. Trypanosome stocks started with feeder layer cells were adapted to axenic conditions within four weeks by transferring trypanosome-containing supernatant to wells without feeder cells. The culture plates were incubated at 37°C in a 4% CO2/air atmosphere.

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Drugs

DL-ct-Difluoromethylornithine hydrochloride monohydrate (DFMO) was kindly sup- plied by the Merrell Dow Research Institute (Cincinnati, OH, U.S.A.), diminazene aceturate by Hoechst AG (F.R.G.) and Mel W (Trimelarsan) by Specia, (France). Isometamidium chloride (Samorin, May & Baker, U.K.) and suramin (Naganol, Bayer AG, F.R.G.) were purchased commercially.

Growth inhibition test

The growth inhibition test was carried out as described by Kaminsky and Zweygarth (1989) with minor modifications. Suspensions of culture-derived trypanosomes were adjusted to a concentration of 2 × 105 ml and 375 ~tl were pipetted into a 48-well culture plate (Costar). An equal volume of 2 x concentrated drug solution in medium was added. Each drug concentration was tested in triplicate, and was repeated at least twice. After 24 h of incubation, 500 p.1 aliquots were removed from each well, transferred into disposable cups (Sarstedt, Nfimbrecht, F.R.G.), fixed with 6 111 for- malin (37%), diluted with Isoton (Coulter Electronics, Nairobi, Kenya) and counted in a Coulter Counter model ZM (70 ~tm aperture). The number of generations in drug-treated cultures was calculated for each well and the relative growth of trypano- some populations was determined by comparison with the number of generations (100%) in control cultures.

Growth inhibition (G!) [%] was calculated as follows:

[log n,E4) (with drug)__-_l_og_n~o}]~ 100 GI = 100 - [log n{24} (controls) - log n{o)] J ×

(nto) = Initial number of trypanosomes; nt24) = number of trypanosomes after 24 h of incubation).

The drug concentration, which inhibited growth of trypanosome populations by 50% (ICso), was determined using logit analysis, minimum chi square method. Values > 100% and ~<0% growth were rejected.

Test for drug synergy

Determination of the effect of drug combinations was done by the method of Beren- baum (1978). Briefly, the IC5o of drugA in combination with a constant amount of druga was expressed as a fractional inhibitory concentration (FIC) of the IC5o value of drugA alone (DrugA FIC = IC5o of drugA in combination/ICso drugA alone). The concentration of drugB which was present in that combination was expressed as the FIC of the IC5o value of drugB alone (DrugB FIC = concentration of drugB in combi- nation/ICso drugB alone). The FICs for drugB with a constant amount of drugA were determined by the same method. The corresponding FIC values were used to con- struct isobolograms. When the isobole is concave the combination is synergistic (potentiation), the combination is antagonistic when it is convex. The points lie on the connecting line joining the two FICs of 1 when the combination is additive.

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In vivo experiments

Swiss mice of either sex, weighing 20-35 g, were used. Groups of eight mice were injected intraperitoneally (i.p.) with 1 x l0 s trypanosomes of the respective stock. Treatment was performed 24 h after infection by i.p. inoculation of the drug solution. Mice were checked three times a week for trypanosomes in their tail blood by the wet blood film technique. They were considered cured when no trypanosomes were detected from 5 to 60 days after the end of the therapy. D F M O was administered in the drinking water as a 2% solution for a period of 3, 5 or 10 days.

Results

No living trypanosomes were observed after 24 h of incubation in medium containing human serum, irrespective of whether human serum was used alone or together with horse serum. Control cultures maintained in medium with horse serum showed nor- mal growth. All stocks and the clone were, therefore, considered as T.b. brucei.

A 2% solution of DFMO given over a five day period cured mice infected with all stocks, except stock CP 1284, where only three out of eight mice were cured. Shortening the treatment period to three days was effective in clone ILTat 1.4, but not in stock CP 547 (Table 3).

The IC50 values for DFMO of the different stocks obtained in vitro are shown in Table 4. Clone ILTat 1.4 was the most susceptible organism (IC5o 81 IxM). The lowest IC5o value for the stocks was 93 ~tM for stock CP 161, which was resistant to diminazene aceturate and isometamidium chloride, but susceptible to suramin in mice. In contrast, the highest IC5o value determined was 691 ixM for stock CP 1284, which was susceptible to diminazene aceturate and suramin, but partially resistant to isometamidium chloride in mice.

Combining DFMO and mel W resulted in an additive effect in vitro in a mel W-susceptible (CP 2137) and in a mel W-resistant stock (CP 2469) (Figs. 1 and 2). Clone ILTat 1.4 showed an antagonistic response to the DFMO-mel W combination (Fig. 3), although it was highly susceptible to D F M O in vivo and in vitro as well as

TABLE 3

Susceptibility of Trypanosoma brucei brucei to D F M O treatment in mice

Trypanosome stock

TreatmenP period (days)

3 5 10

ILTat IA b 8/8 c 8/8 nd CP 547 1/8 8/8 nd CP 1284 nd 0/8 3/8 CP 2107 nd 8/8 nd CP 2469 nd 8/8 nd

aDFMO was given as a 2% solution, blLTat 1.4 is a clone. CResults are expressed as cured/treated mice. nd = experiment not done.

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TABLE 4

Susceptibility of Trypanosoma brucei brucei stocks in vitro to DFMO

Trypanosome IC5o Relative resistance stock (p.M) factoP

ILTat 1.4 b 81 0.9 CP 161 93 1.0 CP 271 238 2.6 CP 547 204 2.2 CP 1154 136 1.5 CP 1184 273 2.9 CP 1284 691 7.4 CP 2107 179 1.9 CP 2137 219 2.4 CP 2469 147 1.6 CP 2711 106 1.1

aThe relative resistance factor was obtained by dividing the ICs0 values by the value of the most susceptible stock CP 161. blLTat 1.4 is a clone.

liMel W FIC

0.75

0.5

0 0.25 0.5 0.75 1 I]FMO FIC

Fig. 1. Isobologram showing an additive effect of DFMO and mel W in stock CP 2137. Control IC5o normalized to 1 unit of ICso refers to DFMO alone (X-axis; FIC = fractional inhibitory concentration) and mel W alone (Y-axis; FIC). The solid line represents the isobole of the drug combination. The dotted

line joining the two FICs of 1 is the isobole of an additive combination.

to m e l W t r e a t m e n t a l o n e ( d a t a n o t s h o w n ) . A n a n t a g o n i s t i c effect w a s o b s e r v e d ,

w h e n c l o n e I L T a t 1.4 w as i n c u b a t e d w i t h D F M O a n d s u r a m i n (Fig . 4). W h e n a

c o m b i n a t i o n o f D F M O a n d s u r a m i n w a s u s e d in t he s u r a m i n - r e s i s t a n t s t o c k C P

2107, a c o n c a v e i s o b o l o g r a m f o r D F M O - s u r a m i n i n d i c a t e d a d i s t i n c t syne rg i s t i c

effect ( F i g u r e 5).

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i Mel W FIC

0.75

0.5

o

0.25

0 0.~ 0.5 075 1 DFM0 FIC

Fig. 2. Isobologram showing an additive effect of DFMO and mel W in stock CP 2469. Control IC5o normalized to 1 unit of ICso refers to DFMO alone (X-axis; FIC = fractional inhibitory concentration) and mel W alone ( Y-axis; FIC). The solid line represents the isobole of the drug combination. The dotted

line joining the two FICs of I is the isobole of an additive combination.

1,75 Mel W FIC

1,5

1.25

1

0.75

0.5

0.25

0125 0mS 0"~ 1 GeMO FIC

I

t25

Fig. 3. lsobologram showing an antagonistic effect of DFMO and mel W in clone ILTat 1.4. Control IC5o normalized to 1 unit of IC~0 refers to DFMO alone (X-axis; F1C = fractional inhibitory concentra- tion) and mel W alone (Y-axis; FIC). The solid line represents the isobole of the drug combination. The

dotted line joining the two FICs of 1 is the isobole of an additive combination.

Discussion

The susceptibility of different stocks of T.b. brucei to DFMO was tested in vitro and in vivo. The stocks exhibited different levels of susceptibility to the drug +in vitro; 7-fold higher concentrations of DFMO were necessary to inhibit the growth of the least susceptible stock (CP 1284) compared with the most susceptible (CP 161). Differences in DFMO susceptibility were not related to growth characteristics, e.g. generation time in culture, nor to their virulence in mice (data not shown). These

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1,5 Suramin FIC

n

0.75

0.5 ....

0.~

o 0.5 o. s i DFMO FIC

i

1.5

Fig. 4. Isobologram showing an antagonistic effect of DFMO and suramin in clone ILTat 1.4. Control IC5o normalized to 1 unit of IC5o refers to DFMO alone (X-axis; FIC = fractional inhibitory concentra- tion) and suramin alone (Y-axis; FIC). The solid line represents the isobole of the drug combination. The

dotted line joining the two FICs of 1 is the isobole of an additive combination.

~. Suramin FIC

0.75

B

0.5 o u

0.25

00 0.~ 015 0.75 I DFMO FIC

Fig. 5. lsobologram showing a potentiation of DFMO and suramin in stock CP 2107. Control IC5o normalized to 1 unit of IC5o refers to DFMO alone (X-axis; FIC = fractional inhibitory concentration

and suramin alone (Y-axis; FIC). The solid line represents the isobole of the drug combination.

results show a high degree of variation of D F M O susceptibility among T.b. brucei field isolates.

These differences in susceptibility to D F M O in vitro were also expressed in vivo. A treatment period of three days was sufficient to cure mice infected with ILTat 1.4 while other stocks required at least a five-day treatment period. Stock CP 1284 was not eliminated from all mice even after a ten-day treatment period.

The combination of suramin and D F M O was synergistic in a suramin-resistant T.b. brueei stock in vitro. Similar results were obtained by McCann et al. (1983) who found that this combination was effective against an acute T.b. brucei infection in mice when either drug alone was ineffective at the dose used for the combined

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treatment. In a mouse model of late-stage African trypanosomiasis (Jennings et al., 1977), Clarkson et al. (1984) showed that the combination of DFMO and suramin acted synergistically. Clone ILTat 1.4 was susceptible to suramin and DFMO in vitro (Kaminsky and Zweygarth, 1989), but the combination of both had an antagonistic effect. This was also observed with mel W and DFMO. It is not possible to explain why these drug combinations reacted antagonistically with clone ILTat 1.4. These results stress the importance of using more than one clone or stock as a reference in chemotherapeutic experiments. The observed differences of synergistic and antagonis- tic effects of drug combinations among parasite stocks or clones suggest that chemo- therapy of trypanosomiasis can be significantly improved if rapid and reliable sensitivity assays are applied (Kaminsky and Zweygarth, 1989; Brun and Kunz, 1989).

The combination of mel W and DFMO showed an additive effect for a mel W-susceptible (CP 2137) and a reel W-resistant stock (CP 2469). This drug interaction was independent of the drug susceptibility pattern of the respective organisms. How- ever, the dose varied with drug susceptibility. A combined treatment scheme reduced the curative dose levels of the drugs under experimental conditions. In contrast to our findings of an additive effect of DFMO and mel W in vitro, Jennings (1988) found a potentiation of a similar combination in mice, using DFMO and the arsenical melarsoprol. This discrepancy might be due to the different stocks used, or to the fact that an antibody response is necessary to eliminate the parasites during DFMO treatment. De Gee et al. (1983) showed that immunosuppression reduced the benefi- cial effects of treatment. The success of treatment also varied with the inherent ability of different mouse strains to mount an antibody response. Therefore an additive effect in vitro may be found to be potentiating in vivo due to the additional influence of the immune response.

The application of DFMO in the drinking water makes it difficult to quantitate the in vivo-effect of drug combinations. Accurate dosages of DFMO cannot be given for individual mice and considerable variations can occur. Romijin et al. (1987) found that the individual uptake of DFMO in the drinking water during a 14 h treatment period ranged between 350 and 2800 mg/kg. They found however, a signi- ficant correlation between the dose of DFMO, which was taken up by each mouse, and the concentration of DFMO in the serum and other tissues.

The variations of susceptibility to DFMO of T.b. brucei stocks reflect their natural resistance since resistance to DFMO shows no cross-resistance to commercial trypan- ocides presently in use. These potential variations in DFMO susceptibility may also occur in other T. brucei spp. and indicate possible complications in DFMO treatment of the Trypanozoon species causing sleeping sickness.

Acknowledgements

The authors would like to thank Mr. R. Farah, Mr. D.E. Ouma, Mr. A.M. Mutavai, and Mr. F. Chuma for their valuable technical help during the experiments. The paper is published by kind permission of the Director of the Kenya Trypanosomiasis Research Institute (KETRI), Dr. A.R. Njogu. DFMO was a generous gift of the Merrell Dow Research Institute (Cincinnati, OH, U.S.A.).

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