Pharmacodynamic interactions among atovaquone, proguanil and cycloguanil against Plasmodium falciparum in vitro

TRANSACTIONSOF THE ROYALSOCIETYOF TROPICALMEDICINEAND HYGIENE(2003) 97, 331-337

Pharmacodynamic interactions among atovaquone, proguanil and cycloguanil against Plasmodium falciparum in vitro Mita M. T h a p a r 1, S e e m a Gupta 1, Carl Spindler 1, Walther H. W e r n s d o r f e r 2 and A n d e r s B j 6 r k m a n a 1Unit of Infectious Diseases, Department of Medicine, Karolinska Hospital, Stockholm, Sweden; 2Department of Specific Prophylaxis and Tropical Medicine, Institute of Pathophysiology, University of Vienna, Austria Abstract Synergistic interaction between atovaquone and proguanil has been suggested as the reason for the effectiveness of Malarone ®. The pharmacodynamic interactions among atovaquone, proguanil and its metabolite cycloguanil were investigated in 4 Plasmodium falciparum parasite strains by culture assays in vitro. The response parameters were determined and 2 statistical methods, log-concentration/response probit method and sum of fractional inhibitory concentrations (ZFIC) method, were used to analyse the experimental data. Within therapeutically relevant concentration ratios, the combination of atovaquone and proguanil showed mean ZFICs of 0.37 at ECs0 (50% effective concentrations) and 0.13 at ECg0, indicating high synergism. The combination of atovaquone and cycloguanil yielded corresponding mean ZFICs of 3.70 and 2.11, indicating antagonism. The EC~0 and ECg0 values for proguanil alone were not influenced by RPMI-1640 medium with low concentrations of paraaminobenzoic acid and folic acid (LPLF culture medium), whereas the ECs0 and ECg0 values for cycloguanil were more than 10 times lower in LPLF medium than in normal RPMI-1640 medium. This confirms the hypothesis that proguanil may act on another target than dihydrofolate reductase. We conclude that the effectiveness of Malarone ® is due to the synergism between atovaquone and proguanil and may not require the presence of cycloguanil. Keywords: malaria, Plasmodium falciparum, chemotherapy, pharmacodynamics, in vitro, atovaquone, proguanil, cycloguanil, Malarone ® Introduction Malarone ® (GlaxoSmithKline, Stevenage, UK), a fixed-dose combination of atovaquone and proguanil, is a recent option for treatment and prophylaxis against Plasmodium falciparum. The effectiveness of the combination appears to be a result of the synergistic interaction between atovaquone and proguanil (Canfield et al., 1995; Edstein et al., 1996) where atovaquone is the main antimalarial compound while proguanil acts as an activity enhancer (Srivastava & Vaidya, 1999). However, the mechanism of synergistic interaction between atovaquone and proguanil as well as the action of proguanil remains unclear. Proguanil has previously been regarded as a pro-drug metabolized to cycloguanil, which is considered to have the main antimalarial activity through the inhibition of dihydrofolate reductase (DHFR) in the pyrimidine biosynthetic pathway. However, proguanil has been shown to have intrinsic antimalarial activity against P. falciparum strains in vitro (Sucharit et al., 1985) including cycloguanil-resistant parasites (Watkins et aL, 1984). This activity appears to be independent of D H F R activity according to recent observations both in vitro (Fidock et al., 1998) and in vivo (Kaneko et al., 1999). Only 1 study has been performed to evaluate the nature of pharmacodynamic interactions between atovaquone and proguanil as well as atovaquone and cycloguanil in vitro (Canfield et al., 1995). The combinations were tested at 4 fixed concentration ratios. The interaction between atovaquone and cycloguanil varied from weak antagonism to weak synergism. However, synergy was observed between atovaquone and proguanil at the 4 tested concentration ratios. The effectiveness of the combination of atovaquone and proguanil against P. falciparum has been tested and confirmed both in vitro (Edstein et al., 1996) and in vivo (Radloff et al., 1996; Anabwani et al., 1999; Looareesuwan et al., 1999). However, data are lacking on pharmacodynamic interactions among atovaquone, proguanil, and cycloguanil in vitro, considering a range of concentration

ratios of the 3 compounds as found in vivo. The role of cycloguanil in the combination is still unclear, including the role of D H F R in the mechanism of action of proguanil. The present study, therefore, investigated interactive profiles of the 3 compounds against 4 different strains ofP. falciparum in vitro using a wide range of concentration ratios with relevance to the situation in vivo. The experiments were performed both in the presence and absence of excess paraaminobenzoic acid (PABA) and folic acid to examine the role of D H F R in the mechanism of action of proguanil and cycloguanil.

Materials and M e t h o d s Drugs and parasites Atovaquone, proguanil, and cycloguanil were obtained from GlaxoSmithKline, Greenford, UK. Four P. falciparum parasite strains were used for all the experiments in vitro, i.e. F32, FCR3, K1, and LS25 originating from Tanzania, Thailand, The Gambia, and Kenya/Uganda, respectively. In our laboratory, all 4 strains showed resistant profiles to chloroquine and partial (F32, FCR3) to full (K1, LS25) resistance to quinine. The study was carried out between 2001 and 2002.

Parasite cultures The parasite strains were kept in continuous culture (Trager & Jensen, 1976) in normal RPMI-1640 medium (Gibco BRL, Life Technologies AB, Sweden). The medium was supplemented with 25 mM HEPES buffer, sodium bicarbonate (2 mg/mL), gentamicin (0.5 gg/mL) and 10% human type AB-positive serum. Uninfected h u m a n type O-positive erythrocytes were used after they were washed twice with Tris Hank's buffer (SBL Vaccine AB, Stockholm, Sweden). The experiments were performed in normal RPMI-1640 culture medium unless otherwise stated where special RPMI- 1640 medium with low concentrations of PABA (0.01 rag) and folic acid (0.5 gg) (LPLF culture medium) was used.

Drug preparation and design of imeraction experiments Address for correspondence: Mira M. Thapar, Malaria Research Unit, M9/02, Karolinska Hospital, SE 17176, Stockholm, Sweden; phone +46 8 51773358, fax +46 8 51776740, e-mail [email protected]

The 3 compounds were dissolved in 95% (v/v) ethanol. The solutions were diluted with ethanol for atovaquone and distilled water for proguanil and cycloguanil to obtain stock solutions of 10 -3 M of the respec-

M.M. THAPARETAL.

332 tive drugs. A series of 10-fold dilutions (10 4-10 11) was prepared from the stock solution. The drug solutions were introduced into 96-well flat-bottomed microtitre plates and dried prior to the culture experiments in vitro. In all experiments, nonsynchronized cultures started with an initial parasitaemia of 0.2-0.5% and haematocrit of 5%. The plates were incubated at 37 °C for 48 h in candle jars. Both the parasite growth and the degree of inhibition were estimated by determination of parasite counts (number of infected RBCs per 10000 RBCs) on Giemsa-stained thin blood films by light microscopy. All the experiments were done in duplicate. Tests were first run for mono-drugs (in duplicate) to assess individual drug sensitivity in all the strains in vitro. The 50% effective concentration (ECs0) values were determined. Drug solutions were then prepared for the drug interaction experiments on the basis of these estimated ECs0 values. T h e concentrations in the final solutions were adjusted to a range between approximately 10-2-102 times the ECs0 values of the respective drugs, using geometric progressions. There were 7 to 8 different concentrations for each drug. A chequerboard design (pattern) was used for the interaction experiments, with a single column for the individual test drugs and duplicate columns for the drug-free controls.

Data analysis Since the response of P. falciparum to the test drugs is log-normal, the statistical analysis based on the logconcentration/response probit method can be used, as described by Litchfield and Wilcoxon (Litchfield, 1949). Drug concentrations were transformed into logarithms (logz) and the percentage inhibition values were transformed into probits (y). The transformed data were processed in a linear least-square regression (Daniel, 1991). The various inhibitory concentrations were calculated according to the formula log× = ( p r o b i t y - a ) / b , followed by taking the anti-log×, where the parameters a and b are the intercept and slope respectively. Growth inhibition at a given drug concentration was calculated according to the formula probit y = a + b log×, entering given logz and converting the resulting y from probit to percent inhibition, using a probit table (Hassan Alin et al., 1999). The assessment of drug interaction was based on 2 methods:

Calculation of observed~expectedECso and ECgo values In this method of assessment of drug interaction, the observed/expected (O/E) ECs0 and EC90 values of the drug combinations were plotted against the given molar drug concentration ratios. The expected concentrations were calculated on the basis of response to the single compounds, assuming fully additive interaction (the combined effect (CE) of the drugs can be expressed by the formula CE = a + [{ 1 - a} × b] where a and b are the proportions of the parasite population inhibited by drug A and B, respectively). An O/E value of more than 2 denotes antagonism, values between 2 and 1 denote partial addition. An O/E value of 1 denotes fully additive activity, while values between 1 and 0.5 denote

low-grade synergism and values below 0.5 indicate higher degree of synergism.

Calculation of sum of fractional inhibitory concentrations This method of assessment of drug interaction is based on the calculation of the sum of fractional inhibitory concentrations (ZFIC) for each drug combination, strain and various ECs, at the given EC (Berenbaum, 1978), using the formula: (ECx of agent A in mixture/ECx of agent A alone) + (ECx of agent B in mixture/ECx of agent B alone). Values ofYFICs < 1 denote synergism, ZFICs 1> 1 and < 2 denote additive interaction, EFICs t> 2 and < 4 denote slight antagonism, and YFICs f>4 denote marked antagonism. Z F I C s < 0 . 5 indicate substantial synergism. These concentrations of various combinations, at ECs0 and ECg0, were plotted in isobolograms and ZFICs < 1 (indicating synergism) appear below the lines drawn between the relevant effective concentrations obtained with the single compounds.

Interpretation in relation to clinically relevant concentration ratios In order to project the observed interactions of the various drug combinations in vitro onto the situation in vivo, special consideration was given to the clinically relevant concentration ranges and ratios for atovaquone, prognanil and cycloguanil achieved during a standard prophylactic regimen. These ratios were derived from the pharmacokinetic concentration-time profile achieved at steady state within 24 h after the daily dose of Malarone ® (Thapar et al., 2002) as shown in Table 1.

Results The basic sensitivity data of the 3 compounds against the 4 P. falciparum strains are presented in Table 2. Table 2 also shows the correlation coefficients and the X2 values for heterogeneity (Litchfield, 1949). All regressions showed satisfactory values for r and X2. For atovaquone, the mean values for ECs0 and EC90 were similar in strains F32, FCR3, and K1 but were approximately 2 orders of magnitude higher in strain LS25. For proguanil, the mean EC90 values were also similar for F32, FCR3, and K1 but were 5 times higher compared with LS25. For cycloguanil, only minor differences were found between F32, FCR3, and K1. The antimalarial activity of proguanil was similar in LPLF and normal RPMI-1640 culture medium whereas the efficacy of cycloguanil was more than 20-fold higher in LPLF than in normal RPMI-1640 medium. Log-concentration-response probit regressions for atovaquone and proguanil in strains F32, FCR-3, and K1 are shown in Fig. 1. The interactive parameters of the combination experiments between the 3 compounds, as determined by ZFICs method, are summarized in Table 3. The interactive profiles obtained from EFICs method are shown as isobolograms in Figs 2 - 4 . Atovaquone and proguanil showed an interactive profile in which synergism and addition prevailed (Table 3, Fig. 2). The (O/E) ECs0 and EC90 values for atovaquone-proguanil combination varied from 7 × 10 -~ to I0 (indicating high

Table 1. Concentrations o f atovaquone (ATO), proguanil (PROG), and cycloguanil (CYCLO) and their ratios achieved during 24 hours after administration of a standard chemoprophylactic single dose o f ATO and PROG (Malarone ®) at steady state Concentration (riM) ATO PROG CYCLO

10271-14196 131-473.5 57-154.5

Concentration ratios (range) PROG/ATO CYCLO/ATO CYCLO/PROG

0.01-0.035 0.005-0.01 0.2- 0.5

DYNAMICS OF ATOVAQUONE, PROGUANIL AND CYCLOGUANIL

333 T h e r e was n o significant difference in the i n t e r a c t i o n between atovaquone and proguanil when experiments were p e r f o r m e d in L P L F c o m p a r e d to n o r m a l R P M I 1640 culture m e d i u m (not s h o w n in the Tables). F o r graphic display of the i n t e r a c t i o n b e t w e e n a t o v a q u o n e

synergism to a n t a g o n i s m ) in all the tested strains. H o w ever, the synergism was h i g h (O/E E C values b e t w e e n 1 × 10 -s a n d 0.1) in all the strains w h e n only c o n c e n t r a t i o n ratios o b s e r v e d w i t h i n t h e t h e r a p e u t i c range were c o n s i d e r e d (only F C R 3 s h o w n here in Fig. 2C). A 99.99 -99.9 • FCR3 K1

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334

M.M. THAPAR E T A L .

Table 2. G r o w t h i n h i b i t i o n of the four s t r a i n s of P l a s m o d i u m f a l c i p a r u m a t o v a q u o n e , p r o g u a n i l , a n d cycloguanil

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Plasmodiumfa~iparumstrain Degree of efficacy

F32 a

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FCR3 a

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23.9 124.4 0.9958 0.0714

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2.672 49.87 0.9902 0.1148

2.51 45.03 0.9795 0.2634

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aCultivated in normal RPMI-1640 medium. bCultivated in special RPMI-1640 medium (LPLF) with low concentrations of paraaminobenzoic acid (0.01 mg) and folic acid (0.5 ~g). °50% effectiveconcentration. d90% effectiveconcentration. eCorrelation coefficient. f)~2for heterogeneity (Litchfield and Wilcoxon).

Table 3. Interactions between atovaquone, proguanil, and cycloguanil against P l a s r n o d i u m f a l c i p a r u m according to parasite strain and effective concentrations Geometric mean (range) ZFIC Parasite strain Atovaquone-proguanil F32 1.60 FCR3 0.37 K1 0.46 LS25 0.48 Atovaquone-cycloguanil F32 0.98 FCR3 3.70 K1 0.94 LS25 1.33 Proguanil- cycloguanil F32 2.27

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SYN-ANT

SYN, synergistic;ADD, additive; SLANT, slightly antagonistic; ANT, antagonistic. ~Calculated by fractional inhibitory concentrations (EFICs) method (Berenbaum, 1978).

and proguanil the O/E system seems to be more appropriate than the ZFIC method as both compounds exhibit rather strong interaction over a wide concentration range. The interaction between atovaqnone and cycloguanil varied from synergism to antagonism. Additive to slightly antagonistic and antagonistic activity prevailed throughout the concentration ratios for FCR3, K1, and LS25 (Table 3 and Fig. 3). The O/E ECso and EC90 values for the atovaquone-cycloguanil combination varied from 1 (additive effect) to 50 (strong antagonism) in all the 4 tested strains. Antagonism prevailed (O/E EC50 and EC90 values between 10 and 50) at concentration ratios within the therapeutic range as shown in Fig. 3C for FCR3. The interaction between proguanil and cycloguanil was tested in 1 strain (F32) only. Synergistic to additive interaction was observed for the wide range of concentration ratios tested (Table 3, Fig. 4). The O/E ECs0 and EC90 values for the proguanil-cycloguanil combination varied from 0.8 to 7, but additive activity pre-

vailed and was also predominant within the range of therapeutic concentration ratios (Fig. 4).

Discussion Atovaquone, an analogue of ubiquinone, acts by inhibiting electron transfer in the mitochondrial respiratory chain at the cytochrome bCl complex (Fry & Pudney, 1992), thereby inhibiting de novo pyrimidine biosynthesis in P. falciparum (Hudson et al., 1991). However, the use of atovaquone alone in the treatment of patients with P. falciparum malaria was not recommended as it resulted in a high rate of recrudescence (Chiodini et al., 1995; Looareesuwan et al., 1996). Instead it was realized that the value of atovaquone was in combination with proguanil because of marked synergism (Canfield et al., 1995; Edstein et al., 1996). Proguanil is partly metabolized to cycloguanil, which acts by inhibiting D H F R in the pyrimidine biosynthetic pathway. However, proguanil has been shown to have some intrinsic antimalarial activity against P. falciparum strains in vitro (Sucharit et al., 1985; Eriksson et al.,

DYNAMICS OF ATOVAQUONE, PROGUANIL AND CYCLOGUANIL A

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Fig. 2. Isobolograms of the interaction between atovaquone (ATO) and proguanil (PROG) against Plasmodium falciparum strain FCR3, at 50% and 90% effective concentrations: (A) ECs0 and (B) EC90. Datum points (triangles) below the lines connecting the ECs0 and ECg0 for ATO and PROG denote synergism. The ECs0 and ECg0 for ATO and PROG are derived from log-probit regressions. (C) Ratios between observed and expected ECs0 and EC90 values for mixtures of ATO and PROG against FCR3. The area between the 2 horizontal lines represents the range of additive activity.

1989) and also some activity against cycloguanil-resistant parasites in vitro (Watkins et al., 1984). We investigated interactions among atovaquone, proguanil and cycloguanil against P. falciparum in vitro. T h e assessment of the drug interaction was based on 2 statistical methods, i.e. calculation of the EFICs and the O/E ECs0 and ECg0 values of the drug combinations. T h e m e a n EFICs provided a qualitative range of the interaction profile ranging from substantial synergism to marked antagonism (ZFIC < 0.5 to > 4) at various ECs. T h e isobolograms obtained from the EFIC m e t h o d showed an interactive profile throughout the range of concentration ratios tested between the 2 drugs. This information was generally confirmed by the O/E ECs0 and ECg0 values for each combination. T h e O/E values, however, also provided specific information about the interaction profile in relation to different drug concentration ratios, e.g. those that are therapeutically relevant.

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Fig. 3. Isobolograms of the interaction between atovaquone (ATO) and cycloguanil (CYCLO) against Plasmodiumfalciparum strain FCR3, at 50% and 90% effective concentrations: (A) ECs0 and (B) ECg0. Datum points (triangles) below the lines connecting the ECs0 and EC90 for ATO and CYCLO denote synergism. The ECs0 and ECg0 for ATO and CYCLO are derived from log-probit regressions. (C) Ratios between observed and expected ECs0 and ECg0 values for mixtures of ATO and CYCLO against FCR3. The area between the 2 horizontal lines represents the range of additive activity.

In our experiments against P. falciparum in vitro with proguanil and cycloguanil alone, a more than 10-fold difference was observed in the EC values for cycloguanil and no change was observed in corresponding values for proguanil w h e n normal R P M I - 1 6 4 0 and L P L F m e d i u m were used. This m a y suggest and support the previous in vitro (Fidock et al., 1998) and in vivo (Kaneko et al., 1999) observations that proguanil acts on a therapeutic target different from that of cycloguanil, independent of the activity against D H F R in P. falciparum. T h e ZFIC values for atovaquone and proguanil combination indicate a profile varying from addition to high synergism, with prevailing synergism. Synergism was further confirmed by the corresponding O/E E C values for the combination. This p h e n o m e n o n is apparently

336

M.M. THAPARETAL.

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Fig. 4. Isobolograms of the interaction between proguanil (PROG) and cycloguanil (CYCLO) against Plasmodium falciparum strain F32, at 50% and 90% effective concentrations: (A) ECs0 and (B) ECg0. Datum points (triangles) below the lines connecting the ECs0 and ECg0 for PROG and CYCLO denote synergism. The EC~0 and ECgo for PROG and CYCLO are derived from log-probit regressions. (C) Ratios between observed and expected ECs0 and ECg0 values for mixtures of PROG and CYCLO against F32. The area between the 2 horizontal lines represents the range of additive activity. caused by a potentiation of the effect of atovaquone by proguanil throughout the concentration range. Most importantly, this interaction was highly synergistic (O/ E < 0.5) at therapeutically relevant concentration ratios of proguanil/atovaquone found in the body after oral administration of the combination. The corresponding ZFIC values for atovaquone and cycloguanil combination showed an interaction varying from synergism to antagonism. This interaction was confirmed to be antagonistic at therapeutically relevant concentration ratios by the O/E EC values. Our results thus generally agree with previous findings in vitro (Canfield

et al., 1995; Edstein et al., 1996) but we can conclude that at clinically relevant drug concentrations and combination ratios, the synergistic effect of atovaquone and proguanil was significant whereas cycloguanil exhibited rather an antagonistic effect. It can therefore be concluded that the high clinical efficacy relies on atovaquone and proguanil and does not depend on the metabolism of proguanil to cycloguanil. However, the lack of knowledge of the mechanism of action of proguanil still precludes an explanation of the mechanism of synergy between atovaquone and proguanil. The results from a study in vitro (Srivastava & Vaidya, 1999) showed that proguanil acts as an activity enhancer to atovaquone but the molecular basis behind this enhancement is not known. It is also clear from our results that only relatively low proguanil concentrations are required for achieving the enhancement of atovaquone activity. Resistance to proguanil/cycloguanil has been associated with point mutations in the dhfr gene (Sirawaraporn et al., 1997) and recently, atovaquone resistance has been associated with mutations in the cytochrome b gene (Korsinczky et al., 2000). The risk for selection of resistance by atovaquone is high (mutants arising in one out of three treated infections), when atovaquone is used on its own (Looareesuwan et al., 1996). It would be expected to be reduced by a factor of 102-103 when combined with proguanil (Olliaro, 2001). However, P. falciparum resistance to the combination has been reported recently (Fivelman et al., 2002; Ffirnert et al., 2003). Therefore, it may be important to perform studies with atovaquone and proguanil in vitro with P. falciparum strains with known cytochrome b mutations and different alterations at the dhfr gene, in order to evaluate the possible involvement of this gene in the observed interaction between atovaquone and proguanil. Atovaquone is considered to be extensively protein bound (> 99.9%, unpublished data). There are always limitations inherent in projecting findings in vitro onto clinical in vivo conditions such as, in this case, overestimating the u n b o u n d fraction of atovaquone available for interaction with proguanil when only 10% serum is used. However, with all such limitations, results of our studies, considering wide range of concentration ratios for both the compounds, seem to conclude that the combination of atovaquone and proguanil is synergistic. Combination therapy, with a concept of combining antimalarial agents with different independent combined mechanisms of action, helps in delaying the development of resistance. Artemisinin derivatives reduce parasite density more rapidly then any other antimalarial drug. They also appear to exhibit a significant gametocytocidal effect, thus minimizing the risk of a resistant mutant to be further transmitted. Therefore, the co-administration of artemisinin derivatives with atovaquone and proguanil combinations may provide an alternative option in antimalarial chemotherapy especially against highly resistant strains of P. falciparurn. There is some indication of synergism between atovaquone and artemisinin against P. falciparum in vitro (Canfield et al., 1995; Gupta et al., 2002). It would thus be interesting to extend interaction studies on the combination effect of atovaquone and proguanil with artemisisin or artemisinin derivatives in vitro and also to assess the efficacy of this triple combination in vivo. Acknowledgements

This work was financially supported by grants from the Swedish International Development Agency (Sida/SAREC). References

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Received 8 August 2002; revised 21 November 2002; accepted for publication 28 November 2002.