AAC Accepts, published online ahead of print on 9 June...

Treatment of murine cerebral malaria by artemisone in combination with 1

conventional anti-malarial drugs: anti-plasmodial effects and immune 2

responses 3

4 5 W. Armand Guiguemde,a Nicholas H. Hunt,b Jintao Guo,b Annael 6

Marciano,c Richard K. Haynes,d Julie Clark,a R. Kiplin Guy,a Jacob 7

Golenserb,c,* 8

9

Department of Chemical Biology and Therapeutics, St Jude Children’s 10

Research Hospital, Memphis, Tennessee, USAa; Department of Pathology 11

and Bosch Institute, The University of Sydney, Sydney, Australiab; 12

Department of Microbiology and Molecular Genetics, The Kuvin Center 13

for the Research of Tropical and Infectious Diseases, The Hebrew 14

University of Jerusalem, Jerusalem, Israelc; Centre of Excellence for 15

Pharmaceutical Sciences, North-West University, Potchefstroom, South 16

Africad 17

18

*Corresponding author. Tel.: 97226758090; fax: 97226757425. 19

E-mail address: [email protected] 20

21

22

The decreasing effectiveness of anti-malarial therapy due to drug 23

resistance necessitates constant efforts to develop new drugs. Artemisinin 24

derivatives are the most recent drugs that have been introduced and are 25

considered the first line of treatment but there are already indications of 26

Plasmodium falciparum resistance to artemisinins. Consequently, drug 27

combinations are recommended for prevention of the induction of 28

resistance. The research here demonstrates the effects of novel 29

combinations of the new artemisinin derivative, artemisone, a recently 30

AAC Accepts, published online ahead of print on 9 June 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.01553-13Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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described 10-alkylamino artemisinin derivative with improved anti-31

malarial activity and reduced neurotoxicity. We here investigate its ability 32

to kill P. falciparum in a high throughput in vitro assay and to protect mice 33

against lethal cerebral malaria caused by P. berghei ANKA when used 34

alone or in combination with established anti-malarial drugs. Artemisone 35

effects against P. falciparum in vitro were synergistic with halofantrine 36

and mefloquine, and additive with 25 other drugs, including chloroquine 37

and doxycycline. The concentrations of artemisone combinations that were 38

toxic against THP-1 cells in vitro were much higher than their effective 39

anti-malarial concentration. Artemisone, mefloquine, chloroquine or 40

piperaquine given individually mostly protected mice against cerebral 41

malaria caused by P. berghei ANKA but did not prevent parasite 42

recrudescence. Combinations of artemisone with any of the other three 43

drugs did completely cure most mice of malaria. The combination of 44

artemisone and chloroquine decreased the ratio of pro-inflammatory 45

(interferon-γ, tumour necrosis factor) to anti-inflammatory (IL-10, IL-4) 46

cytokines in the plasma of P. berghei-infected mice. Thus artemisone in 47

combinations with other anti-malarial drugs might have a dual action, both 48

killing parasites and limiting the potentially deleterious host inflammatory 49

response. 50

51

Keywords: 52

Malaria 53

Plasmodium 54

Cerebral malaria 55

Anti-malarial drugs 56

Drug combinations 57

Inflammation 58

59


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Running title: 60

Artemisone combinations: effects on CM and immunity 61

62

63

The dwindling arsenal of drugs for treatment of malaria and the need for 64

developing and selecting new ones is a continuing problem that has been 65

extensively discussed. Most drugs that recently have been approved for 66

human use are artemisinin derivatives (1). However, some artemisinin 67

derivatives that have been introduced have already encountered various 68

degrees of resistance and consequently have been used in drug 69

combinations (2, 3, 4). 70

Artemisone is a recent artemisinin derivative, a semi-synthetic 10-71

alkylamino artemisinin that can be synthesized from dihydroartemisinin. It 72

is an attractive drug because, in comparison to current artemisinins, it is 73

not neurotoxic. In in vitro screens it elicits no cytotoxicity towards brain 74

stem cell cultures and neurofilaments at concentrations up to 25 μM, and 75

has no effect on the respiratory chain (5, 6). Lack of neurotoxicity was also 76

verified in various animal screens (7). 77

Artemisone was found to be highly effective in culture against 78

Plasmodium falciparum (8), in vivo against murine cerebral malaria (CM) 79

induced by P. berghei ANKA (4) and against P. falciparum in monkeys 80

(9). It has been used in a Phase IIa clinical trial for non-severe malaria in 81

humans (10). Artemisone can cure Toxoplasma gondii (11) and Neospora 82

caninum (12) in animal models. 83

We recently have shown in a mouse model of CM that artemisone could 84

prevent death even when administered at relatively late stages of cerebral 85

pathogenesis. No parasite resistance to artemisone was detected and co-86

administration of artemisone and chloroquine was more effective than 87

monotherapy with either drug, leading to complete cure (4). These results 88


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suggest the use of artemisone for combination therapy. However, a 89

thorough study is needed to establish the efficacy of additional 90

combinations of artemisone with commercially available anti-malarial 91

drugs. For this purpose we used high throughput in vitro screening against 92

P. falciparum and a reliable CM model (P. berghei ANKA in C57Bl mice) 93

for in vivo validation (2). 94

When choosing a multiple testing procedure for screening combinatorial 95

drug libraries, natural products or any compound reservoir, the results 96

suggesting further investigation or rejection of a candidate drug often 97

ignore a possible significant effect on the outcome of treatment following 98

the use of these drugs: attenuation of immune responses may alleviate 99

clinical symptoms that are caused by immunopathology. In this context, 100

various forms of severe malaria including CM are the result of 101

immunopathology (13). Therefore, immunomodulators represent an 102

interesting new approach to CM treatment. Likewise, Fasudyl, a Rho 103

kinase inhibitor, was suggested as an adjunctive therapeutic agent in the 104

management of severe malaria (14, 15). IDR-1018, an adjunctive anti-105

inflammatory peptide, was partially protective against murine CM (16). 106

Moreover, anti-plasmodial drugs, including artemisinins, may affect 107

immune responses, in addition to exerting direct effect on the parasites 108

(17, 18). 109

In view of growing information on parasites resistant to artemisinin 110

derivatives, malaria treatments now recommended by the World Health 111

Organization are artemisinin-based combination treatments (ACT). These 112

are combinations of an artemisinin derivative and another structurally 113

unrelated and more slowly eliminated antimalarials (19). Such pairings 114

might include drugs that are not effective as a monotherapy but are useful 115

in the combination, for example Malarone® (combined atovaquone and 116

proguanil) is considered a useful malaria therapeutic agent. However there 117


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are indications of resistance to malarone® (20), stressing the need for a 118

constant search for both new individual anti-malarial compounds and drug 119

combinations. Consequently, we decided to examine the effects of 120

artemisone, a recently discovered 10-alkylamino- artemisinin, alone and in 121

drug combinations, in P. falciparum cultures and in a mouse malaria 122

model, including an examination of the effect of a representative drug 123

combination on cytokine responses that are relevant to CM induction. 124

125

MATERIALS AND METHOD 126

Parasites. P. berghei ANKA was maintained in vivo by serial transfer of 127

parasitized erythrocytes (PE) from infected to naïve mice. Experimental 128

mice were infected by intraperitoneal (i.p.) injection of 5×104 PE from 129

peripheral blood of infected donor mice, an inoculum that caused fatal 130

experimental cerebral malaria (ECM) in at least 80% of infected 131

C57BL/6 mice. The link between early death and ECM in mouse models 132

has been discussed previously (2, 4): mice that died at a parasitemia of 133

20% or below, with accompanying neurological symptoms and drastic 134

reductions in body weight and temperature, were considered to have died 135

of ECM, which where possible was confirmed by the presence in the 136

CNS of hemorrhages, edema and intravascular leukocyte accumulation 137

upon histopathological analysis. Untreated mice that did not die from 138

ECM went on to succumb to severe anemia and hyperparasitemia, as has 139

been reported in all other cases where mice are resistant to ECM induced 140

by P. berghei ANKA (21, 22). 141

The 3D7 strain of P. falciparum (purchased from the American Type 142

Culture Collection, ATCC) was grown in culture as specified later. 143

Animals. C57Bl/6 mice (Harlan, Jerusalem, Israel; Animal Resources 144

Centre, Perth, Australia) aged 7-8 weeks were used in all experiments, 8 to 145

10 mice per group (as described). The mice were housed under standard 146


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light and temperature conditions and provided with unlimited access to 147

water and food. All experiments were carried out in accordance with 148

institutional guidelines for animal care, by protocols approved by the 149

Animal Ethical Care Committee of The Hebrew University of Jerusalem 150

and in accordance with the guidelines under the Australian Code of 151

Practice for the Care and Use of Animals for Scientific Purposes and 152

approved by the University of Sydney Animal Ethics Committee. 153

Parasitemia was monitored microscopically in thin blood Giemsa stained 154

smears prepared from tail blood. Clinical score was evaluated and used for 155

scoring disease severity (Scoring Chart (4)). Mice were euthanized when 156

they reached a degree of disease severity that would inevitably have led to 157

their death. 158

Histology. Mice were deeply anaesthetized with isofluorane and sacrificed 159

by terminal intracardial perfusion with 10 ml ice-cold PBS. Organs were 160

removed and fixed overnight in 10% (v/v) neutral buffered formalin. 161

Paraffin embedded tissues were cut into 5-7 μm slices, deparaffinated, and 162

stained with hematoxylin and eosin before coverslipping. 163

Drugs. Dihydroartemisinin (DHA) and artesunate were purchased from 164

the Kunming Pharmaceutical Corporation. Artemisone was synthesized 165

from DHA and purified by flash column chromatography, followed by 166

recrystallization according to the procedure previously reported (8). 167

Piperaquine was donated by Cipla Ltd, Mumbai, India. It was dissolved in 168

double distilled water, adjusted to pH 3.5 with HCl and injected in 100 μl. 169

Dimethyl sulfoxide (DMSO) and chloroquine diphosphate were purchased 170

from Sigma-Aldrich, Ltd. All artemisinin derivatives were prepared in 171

DMSO according to the required dosage and administered in a volume of 172

20 μl by intraperitoneal injection. Chloroquine diphosphate (Sigma) was 173

dissolved in PBS and administered in a 50 μl volume by intraperitoneal 174

injection. Mefloquine (Sigma) was dissolved in DMSO and used in the 175


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same way as the artemisinin derivatives. Drug structures have been shown 176

elsewhere (2). Artemisone and chloroquine were injected 6 times, twice on 177

days 6, 7 and 8. Piperaquine was injected once a day due to its longer half-178

life in mice (23). 179

Drug toxicity. Drug toxicity was determined in THP-1 cells (human 180

monocytes, ATCC, USA) as previously described (2) using Alamar blue 181

viability assay in 96-well flat-bottom plates (Nunc). The Alamar blue 182

method has been questioned concerning the use of redox active drugs 183

and/or adherent or fast growing cells. However, in our system where the 184

cells were exposed to the drugs before the addition of the indicator and the 185

cells were not adherent or fast growing, it is unlikely that there is a 186

significant aberration of the results. Percent growth inhibition of the cells 187

was calculated according to the equation: % inhibition = [(Fluorescence 188

control – Fluorescence test) / (Fluorescence control)] x 100. 189

Automated screening of in vitro anti-plasmodial activity. Automated 190

screening has been described elsewhere (2). Briefly, parasites were 191

incubated for 72 h and parasitemia was estimated by using a DNA dye 192

solution (SYBR green). Three-fold serial dilutions resulting in 10 different 193

concentrations were examined. Tests were run in triplicates in two 194

independent runs to determine IC50 against the 3D7 P. falciparum strain 195

for each drug. Synergy or antagonism was determined using Bliss 196

independence (24) and fractional inhibition concentration (FIC50)(25). 197

Drug combination were defined as non-additive when more than one 198

binary combination effect lay outside the predicted effect (Bliss); and 199

when more than one FIC50 was outside the 95% confidence interval of a 200

control FIC50 (using a compound against itself as a control). 201

Analysis of in vitro anti-plasmodial activity. The effects of piperaquine 202

and its combinations with artemisone against P. falciparum were evaluated 203

manually in vitro by a luciferin-luciferase bioluminescence assay (2). 204


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Briefly, we used erythrocytic stages of P. falciparum stably expressing the 205

luciferase gene by the hrp2 promoter from a chromosomal locus (Pf:LUC) 206

in 96 well flat-bottom sterile plates. After 48 hours medium was removed 207

and the erythrocytes lysed by lysis buffer of the Bright-Glo Luciferase 208

Assay System (100µl/well). 50µl of the Bright-Glo substrate was added to 209

each well and the luminescence measured by a luminometer (Fluoroskan 210

Ascent FL, Thermo). 211

ELISA assay. ELISA assays for murine plasma cytokine analysis of 212

interleukin(IL)-10, interferon (IFN)γ, IL-4 and tumor necrosis factor 213

(TNF) were purchased from Biolegend, Israel. C57Bl/6 mice were injected 214

with P. berghei ANKA and treated with different drugs at days 6, 7 and 8 215

post infection (PI). On days 0, 5, 8, and 12 PI, mice were sacrificed and 216

blood samples were collected in heparin for plasma cytokine analysis of 217

IL-10, IFNγ, IL4 and TNF by ELISA assay according to the 218

manufacturer's instructions. 219

Statistics. When comparing parasitemia, p values were calculated using 220

Students t-test; for analysis of survival curves, the Kaplan-Meier test was 221

employed. In both cases, values below 0.05 were considered significant. 222

223

RESULTS 224

Evaluation of drug combinations in P. falciparum. Drug susceptibility 225

assays were performed in P. falciparum cultures, using HTS techniques. 226

To evaluate synergistic combinations, we used two orthogonal methods 227

(FIC50 and Bliss independence) and the combination of artemisone with 228

itself was used to define the additive background (Fig. 1). A summary of 229

the overall results of examining combinations of artemisone with 230

conventional anti-malarial drugs is shown in Fig. 2. Artemisone in 231

combination with most antimalarials currently used in the clinic was 232


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additive, except with halofantrine and mefloquine where synergy was 233

identified. No antagonism was identified. 234

Manual analysis of in vitro anti-plasmodial activity revealed identical 235

results (data not shown). Artemisone-piperaquine combinations that were 236

examined only manually depicted synergistic effect. 237

Cytotoxicity assays. Drug toxicity was determined against THP-1 cells 238

using the Alamar blue viability assay. For positive control (maximal 239

growth inhibition) we used KuRei, a cell inhibitor (26). The assay was 240

performed in triplicates and the standard deviation of the activities was 241

within 10% of the mean for each drug. The IC50 values of artemisone and 242

chloroquine were >282 and 313 nM, respectively; the IC50 values for 243

mefloquine and piperaquine were >12.1 and 1.5 μM, respectively. These 244

data, and the results of drug combinations (Fig. 3), should be compared to 245

the effects of the drugs on P. falciparum cultures (2). The anti-plasmodial 246

effects were evident at much lower concentrations (about 80 fold lower). It 247

is obvious that the drug combinations had no synergistic cytotoxicity. 248

The effects of artemisone combination therapy on P. berghey infected 249

mice. Drug concentrations suitable for in vivo combination experiments 250

were selected based on the HTS screening and previous results (2, 4): 251

those that induced a temporary reduction in parasitemia and clinical score. 252

Combination therapy of infected mice with artemisone and either 253

mefloquine, chloroquine or piperaquine was applied to try to prevent the 254

cerebral symptoms and achieve complete cure. Fig. 4, 5a, 5b depict 255

resulting parasitemias, survival curves, body temperatures, weight and 256

clinical scores. Abrupt decline in temperature and loss of weight were 257

reflecting initial symptoms of the disease that were alleviated by the 258

drugs. However, all mice treated with the individual drugs, despite an 259

initial delay in parasitemia increase, eventually succumbed to the disease. 260

In contrast, the combinations prevented CM, delayed recrudescence and 261


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prevented death in most mice. Artemisone-mefloquine combinations were 262

superior to the individual drugs but not as efficient as artemisone 263

combinations with chloroquine or piperaquine. 264

Histology. Brains were assessed at different intervals post infection (at 265

least four matching animals from each group). The results paralleled the 266

clinical score: in control untreated mice, hemorrhages and intravascular 267

leukocyte accumulation were abundant, but in drug treated animals 268

depicting low clinical score there were no such manifestations (data not 269

shown). 270

Analysis of cytokines involved in development of CM. The purpose of 271

this part of the study was to examine a possible correlation between the 272

outcome of the treatment and the immunological status of the treated 273

animals. We examined the effects of different therapies on the plasma 274

levels of representative cytokines in the malaria-infected mice treated at 275

days 6, 7 and 8 PI with artemisone (2x10mg/kg), chloroquine (2x15mg/kg) 276

or their combination. These concentrations were chosen to allow maximal 277

anti-malarial activity (without approaching toxic levels). TNF and IFNγ 278

represent pro-inflammatory cytokines (Th1 type). IL-4 and IL-10 represent 279

anti-inflammatory cytokines (Th2 type). All injected animals had shown 280

early CM symptoms (coat ruffled, hunched, slight decrease in body weight 281

and temperature) before treatment was started (e.g. ruffled coat, wobbly 282

gait; scoring chart,(4)). Cytokine plasma levels were estimated using 283

ELISA on days 0, 5, 8 and 12 PI. Control groups included uninfected 284

untreated, uninfected treated and infected untreated mice. 285

On day 0 (Fig. 6A), in uninfected untreated mice, the IL-10 level in the 286

plasma was lower than that of the other cytokines. On day 5 (Fig. 6B), in 287

infected untreated mice, the most striking events were a significant 288

increase in TNF and decrease in IL-4 levels, in comparison both with the 289

other cytokines and with their level in the control group on day 0. 290


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On day 8 (Fig. 7), in chloroquine and combination treated mice, the plasma 291

level of the anti-inflammatory cytokine IL-4 was higher than in infected 292

untreated mice. The IL-10 level had increased in all treated groups 293

compared to that in untreated mice, where it was below detection. Plasma 294

IFNγ levels were comparable in all groups, although there was a slightly 295

lower level in the combination treated mice. It is interesting that infected 296

untreated mice displayed much higher TNF in plasma compared to all the 297

treated mice. These results are consistent with the involvement of pro-298

inflammatory cytokines such as IFNγ and TNF in CM. 299

Fig. 8 depicts the plasma cytokine levels on day 12. Higher plasma IL-4 300

was seen in the chloroquine and combination treated mice, compared to 301

the other groups. Artemisone treated mice displayed higher plasma IL-10 302

level compared to the other groups. In the combination treated mice, there 303

was lower plasma IFNγ relative to mice treated by individual drugs, TNF 304

in plasma had declined significantly in all drug treated mice, while in 305

infected untreated mice it rose significantly. 306

307

DISCUSSION 308

Drug combinations are the current strategy in malaria treatment to contain 309

resistance to individual drugs or postpone its induction. Vivas et al. (27) 310

found by using isobolograms obtained with susceptible 3D7 and drug-311

resistant K1 P. falciparum strains at the IC50 level, slight antagonistic 312

trends between artemisone and chloroquine, amodiaquine, tafenoquine, 313

atovaquone or pyrimethamine. Additive to slight synergistic interactions 314

were seen with artemisone and mefloquine, lumefantrine or quinine. In 315

vitro automated screening allows for the examination of multiple drug 316

combinations in order to identify potential partner drugs to be used in the 317

clinic, or to avoid combinations that would be antagonistic (Fig. 1, 2). We 318

tested artemisone in combination with most antimalarials currently used in 319


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the clinic and found that it was additive with all drugs tested except 320

halofantrine and mefloquine, where synergy was identified (Fig. 2). Most 321

importantly, no antagonism was identified, implying that artemisone could 322

be paired with current antimalarials for further in vivo combination studies. 323

We selected combinations of artemisone with mefloquine, piperaquine or 324

chloroquine for in vivo evaluations. This decision was partly based on the 325

in vitro synergistic effect of artemisone with mefloquine and additive 326

effect with piperaquine or chloroquine (Fig. 2), and also our experience 327

where piperaquine or chloroquine, in combination with artemiside (another 328

alkyl-amino-artemisinin) were found additive in vitro, and synergistic in 329

vivo (2). Vivas et al. (27) found slight antagonism between the in vitro 330

effects of artemisone and chloroquine, while we found additivity. The 331

difference in these in vitro results is not great and may have its origin in 332

the different experimental conditions. However, both groups report 333

synergistic in vivo effects. This is important because often in vitro results 334

are not translated into the in vivo domain (28). It is especially interesting 335

to examine artemisone (which is already in clinical trials) in combination 336

with chloroquine. The latter drug played an important role in malaria 337

eradication but it remains attractive because of its low cost, and the 338

possibility that when combined with other drugs, treatment success will be 339

significantly increased. Also, it is desirable to elucidate the use of 340

combination of drugs that are less effective alone because of parasite 341

resistance but are efficient together. A clear example is provided by 342

Malarone that comprises the combination of atovaquone and proguanil 343

(resistance to the individual drugs in this combination has been 344

unequivocally demonstrated). 345

Schmuck et al. (29) suggest that artemisone is embryotoxic. However, in 346

that paper there are no comparative experimental results - it focuses on 347

artemisone alone, ignoring other artemisinin derivatives. In a direct in vivo 348


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comparison of artemisone and artesunate, artemisone was not found 349

neurotoxic in contrast with artesunate (7). Moreover, artesunate in low 350

doses was embryotoxic in rats (30). In addition, a fatal artesunate toxicity 351

was reported in a child (31). Yet, artesunate alone or in drug combinations 352

is considered a first line anti-malarial treatment. Indeed, in a limited study 353

involving pregnant women there was no evidence of artesunate 354

embriotoxicity (32). Artemisone is more effective than artesunate in vitro 355

and against murine models of malaria (4, 27). In our experiments, 356

artemisone combinations with the tested anti-malarial drugs were not 357

toxic, both in vitro (Fig. 3) and in vivo. The highest concentrations of 358

artemisone (10mg/kg), in drug combinations, when injected twice a day 359

for three days, had no visible effects on the mice. Overall, artemisone 360

might be considered for treatment of malaria especially in drug 361

combinations, where toxicity of individual drugs can be reduced. 362

In long term experiments we found that mice treated with the individual 363

drugs, despite an initial delay in parasitemia increase, eventually 364

succumbed to the disease. However, most mice treated with artemisone 365

combination therapy were completely cured (Fig. 4, 5). Vivas et al. (27) 366

described some in vivo artemisone–drug interactions in a rodent model by 367

using the Peter’s four day test. This method is inadequate for estimation of 368

the effect of drug treatment on severe malaria (e.g. CM), where 369

pathogenesis is most pronounced a week or more after infection and 370

pathology (or lack of pathology) is the result of a prolonged innate 371

immune response and early acquired immunity (4, 33). 372

The in vivo experiments were performed in a reliable mouse model of CM 373

(22, 34). The underlying mechanism of CM pathogenesis remains 374

incompletely understood but there is widespread agreement that cytokines 375

(and other components of the immune system) have a crucial role in CM 376


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and severe malaria in general, in mice and in humans. An imbalance 377

between the release of pro-inflammatory and anti-inflammatory cytokines 378

has been associated with the central nervous system dysfunction found in 379

human and experimental CM (35). The current study in C57BL/6 mice 380

infected with P. berghei ANKA aimed to investigate whether the anti-381

plasmodial effect and prevention of CM correlate with an immunological 382

shift, expressed in plasma cytokine levels. The inflammatory cytokines 383

TNF, IFN-γ (Th1-type) and anti-inflammatory IL-4 and IL-10 (Th2-type), 384

were determined in plasma by ELISA. 385

In clinical studies of human CM, elevated serum IFN-γ is seen in acute 386

malaria infection in South-East Asian (36) and African (37) patients. 387

Murine and human studies strongly support a role for IFN-γ and 388

downstream immune system processes in the pathogenesis of CM (38, 39, 389

40). Evidence suggesting that TNF is another key element in the 390

pathogenesis of experimental CM has been reviewed extensively (41, 42). 391

This includes the observation of high serum levels of TNF at the onset of 392

CM (43) and the prevention of the neurological syndrome when TNF 393

levels are low (44, 45). Anti-inflammatory cytokines such as IL-10 seem to 394

have a host-protective role in murine malaria. For example, the clinical 395

scores of IL-10 deficient, infected mice were significantly higher when 396

compared with WT mice. In addition, in a susceptible mouse strain, 397

administration of IL-10 gave some degree of protection against CM 398

induced by P. berghei ANKA (46, 47). IL-4 is an anti-inflammatory 399

cytokine. Changes in plasma IL-4 have been reported to correlate with 400

severe malaria (48) but, conversely, increased levels of IL-4 have been 401

linked with reduced immunopathological symptoms (49, 50). 402

Anti-malarial drugs may induce immunological alterations in treated 403

patients and animals. Artemisinins can produce immunosuppression by 404


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down regulating various cytokines of both the innate and acquired immune 405

systems. They induce an anti-inflammatory effect, neutropenia, and 406

reduction in macrophage number and functions, which may produce 407

immunosuppression. Artemisinins also have the ability to induce 408

immunosuppression by inhibiting delayed-type hypersensitivity, 409

lymphocyte proliferation and a rise in antibody level (51). Immune 410

deviation caused by chloroquine and other anti-malarial drugs has been 411

reported (52, 53). 412

We found that the plasma levels of the pro-inflammatory cytokines TNF 413

and IFN-γ increased in P. berghei ANKA-infected mice (Fig. 6 - 8). In 414

parallel with CM reduction, there was an attenuated increase in TNF 415

plasma levels in P. berghei ANKA-infected mice, after treatment with 416

chloroquine, artemisone or the combination of the two. Reduction of IFN-417

γ was achieved with the drug combination. These results agree with the 418

hypothesis that the CM syndrome is a result of a shift in the balance of 419

Th1/Th2 responses toward Th1. While many immune components 420

(cytokines, chemokines, effector cells) and metabolic pathways are 421

involved in processes leading to the expression of CM, one drug may 422

affect only some of these components while another one may affect others. 423

Thus, judicious selection of combination therapy may reduce parasitemia 424

by direct actions on the parasite and also inhibit the severe symptoms of 425

malaria through immunomodulatory actions. In general, all drug treatments 426

reduced inflammatory cytokines and increased anti-inflammatory 427

cytokines (positive effects). However, when examining carefully the data 428

of day 8 and 12 post infection, the positive effects induced by the drug 429

combination was more pronounced (Fig. 6-8; Table 1). 430

Overall, experiments are needed to determine how drugs that are 431

used in combination influence each other in terms of immunomodulation, 432


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toxicity, pharmacokinetic, pharmacodynamic and even pharmacogenetic 433

aspects. 434

435

ACKNOWLEDGEMENTS 436

This work was supported by a grant from the Australian National 437

Health and Medical Research Council (to NH), the Sir Zelman 438

Cowen Universities Fund, the Israel Science Foundation and the 439

Deutsche Forschungsgemeinschaft (DFG) (to JG). We thank Cipla 440

Ltd, Mumbai, India for the kind donation of piperaquine. 441

442

REFERENCES 443

1. Dondorp, A.M., Yeung, S., White, L., Nguon, C., Day, N.P., Socheat, D., von 444 Seidlein, L. 2010. Artemisinin resistance: current status and scenarios for containment. 445 Nat Rev Microbiol. 8, 272-280. 446 447 2. Guo, J., Guiguemde, A.W., Bentura-Marciano, A., Clark, J., Haynes, R.K., 448 Chan, W.C., Wong, H.N., Hunt, N.H., Guy, R.K., Golenser, J. 2012. Synthesis of 449 artemiside and its effects in combination with conventional drugs against severe murine 450 malaria. Antimicrob Agents Ch. 56, 163-173. 451 452 3. Phyo, A.P., Nkhoma, S., Stepniewska, K., Ashley, E.A., Nair, S., McGready, R., 453 ler Moo, C., Al-Saai, S., Dondorp, A.M., Lwin, K.M., Singhasivanon, P., Day, 454 N.P., White, N.J.,Anderson, T.J., Nosten, F. 2012. Emergence of artemisinin-455 resistant malaria on the western border of Thailand: a longitudinal study. Lancet 379, 456 1960-1966. 457 458 4. Waknine-Grinberg, J.H., Hunt, N., Bentura-Marciano, A., McQuillan, J.A., 459 Chan, H.W., Chan, W.C., Barenholz, Y., Haynes, R.K., Golenser, J. 2010a. 460 Artemisone effective against murine cerebral malaria. Malaria J. 9, 227. 461 462 5. Schmuck, G., Haynes, R.K. 2000. Establishment of an in vitro screening model for 463 neurodegeneration induced by antimalarial drugs of the artemisinin-type. Neurotox 464 Res. 2, 37-49. 465 466 6. Schmuck, G., Roehrdanz, E., Haynes, R.K., Kahl, R. 2002. Neurotoxic mode of 467 action of artemisinin. Antimicrob Agents Ch. 46, 821-827. 468 469 7. Schmuck, G.; Temerowski, M.; Haynes, R. K.; Fugmann, B. 2003. Identification 470 of a non-neurotoxic artemisinin derivative in vivo and in vitro. Antimicrob Agents Ch. 471 3, 35-47 472 473


.org/D

ownloaded from

http://aac.asm.org/

8. Haynes, R.K., Fugmann, B., Stetter, J., Rieckmann, K., Heilmann, H.D., Chan, 474 H.W., Cheung, M.K., Lam, W.L., Wong, H.N., Croft, S.L., Vivas, L., Rattray, L., 475 Stewart, L., Peters, W., Robinson, B,L,, Edstein, M.D., Kotecka, B., Kyle, D.E., 476 Beckermann, B., Gerisch, M., Radtke, M., Schmuck, G., Steinke, W., Wollborn, 477 U., Schmeer, K., Römer, A. 2006. Artemisone - A highly active antimalarial drug of 478 the artemisinin class. Angew Chem-Int Edit. 45, 2082-2088. 479

9. Obaldia, N. 3rd, Kotecka, B.M., Edstein, M.D., Haynes, R.K., Fugmann, B., 480 Kyle, D.E., Rieckmann, K.H. 2009. Evaluation of artemisone combinations in Aotus 481 monkeys infected with P. falciparum. Antimicrob Agents Ch. 53, 3592-3594. 482

10. Nagelschmitz, J., Voith, B., Wensing, G., Roemer, A., Fugmann, B., Haynes, 483 R.K., M. Kotecka, B.M., Karl H. Rieckmann, and Edstein, M.D. 2008. First 484 assessment in humans of the safety, tolerability, pharmacokinetics, and ex vivo 485 pharmacodynamic antimalarial activity of the new artemisinin derivative artemisone. 486 Antimicrob Agents Ch. 52, 3085–3091. 487 488 11. Dunay, R., Chan, W.C., Haynes, R.K., Sibley, L.D. 2009. Artemisone and 489 artemiside control acute and reactivated toxoplasmosis in a murine model. Antimicrob 490 Agents Ch. 53, 4450–4456. 491 492 12. Mazuz, M.L., Haynes, R., Shkap, V., Fish, L., Wollkomirsky, R., Leibovich, B., 493 Molad, T., Savitsky, I., and Golenser, J. 2012. Neospora caninum: in vivo and in 494 vitro treatment with artemisone. Vet Parasitol. 187, 99-104. 495 496 13. Grau, G.E., Craig, A.G. 2012. Cerebral malaria pathogenesis: revisiting parasite 497 and host contributions. Future Microbiol. 7, 291-302. 498 499 14. Taoufiq, Z., Gay, F., Balvanyos, J., Ciceron, L., Tefit, M., Lechat, P., Mazier, 500 D. 2008. Rho kinase inhibition in severe malaria: thwarting parasite-induced collateral 501 damage to endothelia. J Infect Dis. 197, 1062-1073. 502 503 15. Waknine-Grinberg, J.H., McQuillan, J.A., Hunt, N., Ginsburg, H., Golenser, 504 J. 2010b. Modulation of cerebral malaria by fasudil and other immune-modifying 505 compounds. Exp Parasitol. 125, 141-146. 506 507 16. Achtman, A.H., Pilat, S., Law, C.W., Lynn, D.J., Janot, L., Mayer, M,L, Ma, 508 S., Kindrachuk, J., Finlay, B.B., Brinkman, F.S., Smyth, G.K., Hancock, R.E. 509 Schofield, L. 2012. Effective adjunctive therapy by an innate defense regulatory 510 peptide in a preclinical model of severe malaria. Sci Transl Med. 4, 135ra64. 511 512 17. Li, T., Chen, H., Wei, N., Mei, X., Zhang, S., Liu, D.L., Gao, Y., Bai, S.F., Liu, 513 X.G., Zhou, Y.X. 2012. Anti-inflammatory and immunomodulatory mechanisms of 514 artemisinin on contact hypersensitivity. Int immunopharmacol. 12, 144-150. 515 516 18. Wang, Z., Qiu, J., Guo, T.B., Liu, A., Wang, Y., Li, Y., Zhang, J.Z. 2007. Anti-517 inflammatory properties and regulatory mechanism of a novel derivative of artemisinin 518 in experimental autoimmune encephalomyelitis. J Immunol. 179, 5958-5965. 519 520


.org/D

ownloaded from

http://aac.asm.org/

19. Nosten, F , White, N.S. 2007. Artemisinin based combination treatment of 521 falciparum malaria. J. Am J Trop Med Hyg. 77, 181-192.31. 522

523 20. Farnert, A., Lindberg, J., Gil, P., Swedberg, G., Berqvist, Y., Thapar, M.M., 524 Lindegardh, N., Berezcky, S., and Bjorkman, A. 2003. Evidence of Plasmodium 525 falciparum malaria resistant to atovaquone and proguanil hydrochloride: case reports. 526 BMJ., 326, 628-629. 527 528 21. Engwerda, C., Belnoue, E., Gruner, A.C., Renia, L. 2005. Experimental models 529 of cerebral malaria, p 103-143. In Langhorne, J. (Ed.), Immunology and 530 Immunopathogenesis of Malaria. Springer-Verlag, Berlin. 531 532 22. Neill, A.L., and Hunt, N.H. 1992. Pathology of fatal and resolving P. berghei CM 533 in mice. Parasitology 105, 165-175. 534 535 23. Moore, B.R., Ilett, K.F., Page-Sharp, M., Jeffrey D. Jago, J.D., Batty, K.T. 536 2009. Piperaquine pharmacodynamics and parasite viability in a murine malaria model. 537 Antimicrob Agents Ch. 53, 2707–2713. 538 539 24. Bliss, C.I. 1999. The toxicity of poisons applied jointly. Ann Appl Biol. 26, 585-540 615. 541 542 25. Canfield, C.J., Pudney, M., Gutteridge, W.E. 1995. Interactions of atovaquone 543 with other antimalarial drugs against Plasmodium falciparum in vitro. Exp Parasitol. 544 80, 373-381. 545 546 26. Reichwald, C., Shimony, O., Dunkel, U., Sacerdoti-Sierra, N., Jaffe, C.L., and 547 Kunick, C. 2008. 2-(3-aryl-3-oxopropen-1-yl)-9-tert-butyl-paullones: a new 548 antileishmanial chemotype. J. Med Chem. 51, 3659-3665. 549 550 27. Vivas, L., Rattray, L., Stewart, L.B., Robinson, B.L., Fugmann, B., Haynes, 551 R.K., Peters, W., Croft, S.L. 2007. Antimalarial efficacy and drug interactions of the 552 novel semi-synthetic endoperoxide artemisone in vitro and in vivo. J Antimicrob 553 Chemother. 59, 658-665. 554 555 28. Golenser, J. Hunt, N. 2013. Neglected Aspects of Drug Discovery - 556 Microbiological Aspects. Curr Clin Pharmacol. 1, 73-80 557 558 29. Schmuck, G., Klaus, A.-M., Krotlinger, F., Langewische, F.W. 2009. 559 Developmental and Reproductive toxicity studies on artemisone. Birth Defects 560 Research. 86, 131–143. 561 562 30. Chung, M-K., Yu, W-J., Lee, J-S., Lee, J-H.. 2013. Embryotoxicity and 563 toxicokinetics of the antimalarial artesunate in rats. Toxicol Res. 29, 27-34. 564 565 31. Camposa, S., de la Cerdac,P., Rivera. A. 2008. Fatal artesunate toxicity in a 566 child. J. Pediatric Infec Dis. 3, 69–75. 567 568 32. McGrady, R., Cho, T., Keo, N.K., Thwai, K.L., Villegas, L., Looareesuwan, 569 S., White, N.J., Nosten, F. 2001. Artemisinin antimalarials in pregnancy: a 570


.org/D

ownloaded from

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prospective treatment study of 539 episodes of multidrug-resistant Plasmodium 571 falciparum. Clin Infect Dis. 33, 2009–2016. 572 573 33. Hunt, N.H., Golenser. J., Chan-Ling, T., Parekh, S., Rae, C., Potter, S., 574

Medana, I.M., Miu, J., Ball, H.J. 2006. Immunopathogenesis of cerebral malaria. Int 575

J Parasitol. 36, 569-582. 576

34. Hunt, N.H., Grau, G.E., Engwerda, C., Barnum, S.R., van der Heyde, H., 577 Hansen, D.S., Schofield, L., and Golenser, J. 2010. Murine cerebral malaria: the 578 whole story. Trends Parasitol. 26, 272-274. 579

35. Torre, D., Speranza, F., Giola, M., Matteelli, A., Tambini, R., Biondi, G. 2002. 580 Role of Th1 and Th2 cytokines in immune response to uncomplicated Plasmodium 581 falciparum malaria. Clin Diagn Lab Immun. 9, 348-351. 582 583 36. Ho, M,, Sexton, M.M., Tongtawe, P., Looareesuwan, S., Suntharasamai, P., 584 Webster H.K. 1995. Interleukin-10 inhibits tumor necrosis factor production but not 585 antigen-specific lymphoproliferation in acute Plasmodium falciparum malaria. J Infect 586 Dis. 172, 838-844. 587 588 37. Ringwald, P., Peyron, F., Vuillez, J.P., Touze J.E., LeBras, J., Deloron, P. 589 1991. Levels of cytokines in plasma during Plasmodium falciparum malaria attacks. J 590 Clin Microbiol. 29, 2076-2078. 591 592

38. Grau GE, Heremans H, Piguet PF, Pointaire P, Lambert PH, Billiau A, 593 Vassalli P. 1989a. Monoclonal antibody against interferon gamma can prevent 594 experimental cerebral malaria and its associated overproduction of tumor necrosis 595 factor. Proc Natl Acad Sci USA. 86, 5572-5574. 596

39. Sanni, L,A,, Thomas, S,R,, Tattam, B,N,, Moore, D,E,, Chaudhri, G, Stocker, 597 R., Hunt, N.H. 1998. Dramatic changes in oxidative tryptophan metabolism along the 598 kynurenine pathway in experimental cerebral and noncerebral malaria. Am J Pathol. 599 152, 611-619. 600 601 40. Yanez, D.M., Manning, D.D., Cooley, A.J., Weidanz, W.P., van der Heyde, 602 H.C. 603 1996. Participation of lymphocyte subpopulations in the pathogenesis of 604 experimental murine cerebral malaria. J. Immunol. 157, 1620–1624. 605 606 41. Clark, I.A., Rockett, K.A. 1994. The cytokine theory of human cerebral malaria. 607 Parasitol Today. 10, 410–412. 608 609 42. Grau, G.E., Piguet, P.F., Vassali, P., Lambert, P.H. 1989b. Tumor necrosis 610 factor and other cytokines in cerebral malaria: experimental and clinical data. Immunol 611 Rev. 112, 49-70. 612


.org/D

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43. Lyke, K.E., Burges, R., Cissoko, Y., Sangare, L., Dao, M., Diarra, I., Kone, A., 613 Harley, R., Plowe, C.V., Doumbo, O.K., Sztein, M.B. 2004. Serum levels of the 614 proinflammatory cytokines interleukin-1 beta (IL-1β), IL-6, IL-8, IL-10, tumor 615 necrosis factor alpha, and IL-12(p70) in malian children with severe Plasmodium 616 falciparum malaria and matched uncomplicated malaria or healthy controls. Infect. 617 Immun. 72, 5630-5637. 618

44. de Miranda, A.S., Lacerda-Queiroz, N., de Carvalho Vilela, M., Rodrigues, 619 D.H., Rachid, M.A., Quevedo, J., Teixeira, A.L. 2011. Anxiety-like behavior and 620 proinflammatory cytokine levels in the brain of C57BL/6 mice infected with 621 Plasmodium berghei (strain ANKA). Neurosci Lett. 491, 202-206. 622

45. Rudin, W., Eugster, H.P., Bordmann, G., Bonato, J., Müller, M., Yamage, M., 623 Ryffel, B. 1997. Resistance to cerebral malaria in tumor necrosis factor-alpha/beta-624 deficient mice is associated with a reduction of intercellular adhesion molecule-1 up-625 regulation and T helper type 1 response. Am J Pathol. 150, 257-266. 626

46. Golenser J., McQuillan J., Hee L., Mitchell A.J., Hunt NH. 2006. Conventional 627 and experimental treatment of cerebral malaria. Int J Parasitol. 36, 583-593. 628

47. Kossodo,S., Monso, C., Juillard P., Velu, T., Goldmani, M., Grau, G.E. 1997. 629 Interleukin-10 modulates susceptibility in experimental cerebral malaria. Immunology 630 91,536-540. 631

48. Cabantous, S., Poudiougou, B., Oumar, A.A., Traore, A., Barry, A., Vitte, J., 632 Bongrand, P., Marquet, S., Doumbo, O., Dessein, A.J. 2009. Genetic evidence for 633 the aggravation of Plasmodium falciparum malaria by interleukin 4. J Infect Dis. 200, 634 1530-1539. 635 636 49. Angulo, I., and Fresno, M. 2012. Cytokines in the pathogenesis of and protection 637 against malaria. Clin Vaccine Immunol. 9, 1145-1152. 638 639 50. de Kossodo, S., Grau, G.E. 1993. Profiles of cytokine production in relation with 640 susceptibility to cerebral malaria. J Immunol. 151, 4811-4820. 641 642 51. Shakir, L Hussain, M., Javeed, A., Ashraf, M., Riaz, A. 2011. Artemisinins and 643 immune system. Eur J Pharmacol. 668, 6-14. 644 645 52. Fryauff, D.J., Church, L.W., Richards, A.L., Widjaja, H., Mouzin, E., 646 Ratiwayanto, S., Hadiputranto, H., Sutamihardja, M.A., Richie, T.L., Subianto, 647 B.,Tjitra, E., Hoffman, S.L. 1997. Lymphocyte response to tetanus toxoid among 648 Indonesian men immunized with tetanus-diphtheria during extended chloroquine or 649 primaquine prophylaxis. J. Infect Dis. 176, 1644-1648. 650 651 53. Ramos-Avila, A., Ventura-Gallegos, J.L., Zentella-Dehesa, A., Machuca-652 Rodriguez, 653 C., Moreno-Altamirano, M.M., Narvaez, V., Legorreta-Herrera, M. 2007. 654 Immunomodulatory role of chloroquine and pyrimethamine in Plasmodium yoelii 655 17XL infected mice. Scand J Immunol. 65, 54-62. 656 657

658


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Figure legends 659

660

FIG 1. Activity per pair of drug combinations. 661

Artemisone pairwise combinations were examined in vitro. Artemisone 662

combined with itself was used as a control for additivity. (A) Fractional 663

IC50 of artemisone in combination with itself. The surface between the red 664

and the blue lines defines the additivity area. The distance between these 665

two lines equals 95% of the FIC50 confidence interval. (B) Heat map 666

representing the percentage of the growth inhibition of the tested drug 667

combinations. (C) Heat map representing the Bliss differential of the drug 668

combinations, the difference in growth inhibition between observed and 669

predicted values. (D) Zbliss is derived from data shown in panel c using Z 670

scores, considering all Bliss differential values as additive values. Values 671

in panels b, c, and d are averages from four dependent replicates. 672

673

FIG 2. Artemisone pairwise combinations examined in vitro 674

Color coded summary of the pairwise combinations examined by the high 675

throughput system. For each pair, FIC50s and Bliss values were calculated 676

(as shown in Figure 3) and the degree of synergy was estimated according 677

to defined criteria based on Z-scores (Zs). 678

679

FIG 3. Cytotoxicity assays of artemisone combinations with standard 680

antimalarial drugs, chloroquine (Ch), mefloquine (Mef) and 681

piperaquine (P). 682

Drug toxicity was determined against THP-1 cells using Alamar blue 683

viability assay. The numbers in the columns represent the experimental 684

results (average of three). The numbers in brackets are the theoretical 685

additive values. For example, artemisone 1 (Art1, 25nM), chloroquine 2 686

(Chl2, 110nM) and their combination inhibit 13%, 28%, and 38% of 687


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untreated THP-1 growth, respectively. The theoretical additive value is 688

41% (13+28). 689

Compound concentrations in the experiment (*nM, **mM): 690

Art1 25*, Art2 75*, Art3 225* 691

Ch1 37*, Ch2 110*, Ch3 330* 692

Mef1 1.3**, Mef2 4.0**, Mef3 12.0** 693

P1 0.38**, P2 0.75**, P3 1.50** 694

695

FIG 4. Parasitemias of infected mice, treated with artemisone or 696

mefloquine or their combinations. 697

Each line represents a single mouse. Infected mice were treated twice a 698

day on days 6-8 post infection. N = 8/group. 699

700

FIG 5. Effect of artemisone, chloroquine and piperaquine on P. 701

berghei ANKA infection. 702

Infected mice were treated on days 6-8 post infection with artemisone (art; 703

twice each day) and chloroquine (chl; twice each day) or artemisone (twice 704

each day) and piperaquine (piper; once per day). 705

5a. Data are parasitemias and survival curves of infected mice. 706

5b. Data are clinical score, weight, and body temperature of infected mice. 707

Values are mean ± SD; n = 8-10/group. 708

709

FIG 6. Plasma cytokine levels in control (A) and infected untreated 710

mice on day 5 (B). 711

Columns and vertical bars represent mean ± SD; n = 8-10/group. 712

713

FIG 7. Effect of artemisone and chloroquine on 714

cytokines in infected mice on day 8 post infection 715


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Plasma cytokine levels in mice treated on days 6-8 (twice each day) 716

post infection by artemisone (Art), chloroquine (Chl) or their 717

combination. Columns and vertical bars represent mean ± SEM; n = 718

8-10/group. 719

720

FIG 8. Effect of artemisone and chloroquine on cytokines 721

in P. berghei ANKA infection on day 12 post infection 722

Plasma cytokine levels in mice treated on days 6-8 (twice each day) 723

post infection by artemisone (Art), chloroquine (Chl) or their 724

combination. Columns and vertical bars represent mean ± SEM; n = 725

8-10/group. 726


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A BFigure 1

C D


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FIG 1. Activity per pair of drug combinations. A t i i i bi ti i d i it A t iArtemisone pairwise combinations were examined in vitro. Artemisone combined with itself was used as a control for additivity. (A) Fractional IC50 of artemisone in combination with itself. The surface between the red and the blue lines defines the additivity area. The distance between ythese two lines equals 95% of the FIC50 confidence interval. (B) Heat map representing the percentage of the growth inhibition of the tested drug combinations. (C) Heat map representing the Bliss differential of the drug combinations the difference in growth inhibition betweenthe drug combinations, the difference in growth inhibition between observed and predicted values. (D) Zbliss is derived from data shown in panel c using Z scores, considering all Bliss differential values as additive values. Values in panels b, c, and d are averages from four p gdependent replicates.


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Test drugFigure 2

AdditiveAdditiveSynergistic: one drug combinations has a ZsBlis differential activity >3 and ZsFIC>3


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FIG 2. Artemisone pairwise combinations examined in vitro. Color coded summary of the pairwise combinations examined by theColor coded summary of the pairwise combinations examined by the high throughput system. For each pair, FIC50s and Bliss values were calculated (as shown in Figure 3) and the degree of synergy was estimated according to defined criteria based on Z-scores (Zs). on June 29, 2018 by guest

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Figure 3.

13 13


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FIG 3. Cytotoxicity assays of artemisone combinations with standardFIG 3. Cytotoxicity assays of artemisone combinations with standard antimalarial drugs, chloroquine (Ch), mefloquine (Mef) and piperaquine (P).Drug toxicity was determined against THP-1 cells using Alamar blue viability assay. The numbers in the columns represent the experimental results (average of three). The n mbers in brackets are the theoretical additi e al es For e ampleThe numbers in brackets are the theoretical additive values. For example, artemisone 1 (Art1, 25nM), chloroquine 2 (Chl2, 110nM) and their combination inhibit 13%, 28%, and 38% of untreated THP-1 growth, respectively. The theoretical additive value is 41% (13+28).( )Compound concentrations in the experiment (*nM, **mM):Art1 25*, Art2 75*, Art3 225*Ch1 37*, Ch2 110*, Ch3 330*Mef1 1 3** Mef2 4 0** Mef3 12 0**Mef1 1.3 , Mef2 4.0 , Mef3 12.0P1 0.38**, P2 0.75**, P3 1.50**


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DMSO control

20

30

Mefloquine 2mg/kg

40

60Figure 4.

0 2 4 6 8 100

10

20

0

20

40

0 2 4 6 8 10

Artemisone 2.5mg/kg

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mia

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05 10 15 20 25 30

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FIG 4. Parasitemias of infected mice, treated with artemisone orFIG 4. Parasitemias of infected mice, treated with artemisone or mefloquine or their combinations.Each line represents a single mouse. Infected mice were treated twice a day on days 6-8 post infection. N = 8/group. Mortality and survival results

i d i t bl 1are summarized in table 1.


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Figure 5a


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FIG 5. Effect of artemisone, chloroquine and piperaquine on P. bergheiANKA infection.

f fInfected mice were treated on days 6-8 post infection with artemisone (art; twice each day) and chloroquine (chl; twice each day) or artemisone (twice each day) and piperaquine (piper; once per day).5a. Data are parasitemias and survival curves of infected mice.5a. Data are parasitemias and survival curves of infected mice.5b. Data are clinical score, weight, and body temperature of infected mice.Values are mean ± SD; n = 8-10/group.


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Figure 5b

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FIG 5. Effect of artemisone, chloroquine and piperaquine on P. bergheiANKA infection.Infected mice were treated on days 6-8 post infection with artemisoneInfected mice were treated on days 6 8 post infection with artemisone (art; twice each day) and chloroquine (chl; twice each day) or artemisone (twice each day) and piperaquine (piper; once per day).5a. Data are parasitemias and survival curves of infected mice.5b D t li i l i ht d b d t t f i f t d5b. Data are clinical score, weight, and body temperature of infected mice.Values are mean ± SD; n = 8-10/group.


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Figure 6g

Average serum cytokines levels on day 0 PI in untreated uninfected group

300ml)

IL 4

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Average serum cytokines levels on day 5 PI in infected untreated group

1600ml)

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FIG 6. Plasma cytokine levels in control (A) and infected untreated y ( )mice on day 5 (B).Columns and vertical bars represent mean ± SD; n = 8-10/group.


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Figure 7

Average IL4 on day 8 PI

700

800Infected un

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Average IL-4 on day 8 PI

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Art 2x10mg/kg

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1000

500

Chl 2x15mg/kg

Art & Chl

0200400600 untreated

Art 2x10mg/kg significant vs. 600

400

200 *Significant vs. untreated

Chl 2x15mg/kg

Art & Chl* * **

0s t&C

0 00


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FIG 7. Effect of artemisone and chloroquine on cytokines in infected mice on day 8 post infection. y pPlasma cytokine levels in mice treated on days 6-8 (twice each day) post infection by artemisone (Art), chloroquine (Chl) or their combination. Columns and vertical bars represent mean ± SEM; n = 8-10/group.


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Average IL10 on day 12 PIAverage IL4 on day 12 PIFigure 8

g y

16000

20000

20000

16000

Average IL4 on day 12 PI

500

600

Untreated uninfected

Treated uninfected

Untreated infected

/ml)

/ml)

/ml)

/ml)

/ml)

/ml)

g/m

l)

Average IL-4 on day 12 PI Untreated uninfected

Treated uninfected

Untreated infected


600

500

4000

8000

12000 * * ***

ml)

12000

8000

4000l)

100

200

300

400

* * **

Art 2x10mg/kg

Chl 2x15mg/kg

Art & Chl

*Significant vs Art &

Conc

entr

atio

n (p

g/Co

ncen

trat

ion

(pg/

Conc

entr

atio

n (p

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ncen

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(pg/

Conc

entr

atio

n (p

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ncen

trat

ion

(pg/

ncen

trat

ion

(pg

Art 2x10mg/kg

Chl 2x15mg/kg

Art & Chl

*Significant vs.

400

300

200

100ml)

** * * *

* * * *0

4000

ratio

n (p

g/m

0

atio

n (p

g/m

l


0

100g

Chl or Chl treatedCon

Average INFγ on day 12 PIU t t dUntreated uninfected

Average IFNγ on day 12 PI

Significant vs. Art treated

100

0

tratio

n (p

g/m

Average TNF on day 12 PIUntreated uninfected

Con

cent

rC

once

ntra

*600

800

1000

120015001800

Untreateduninfected Treated

i f t d*

Untreated uninfected

Treated uninfected

Untreated infected

Art 2x10mg/kg

on (p

g/m

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(pg/

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on (p

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(pg/

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on (p

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(pg/

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n (p

g/m

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1500

1200

Con

cent

600

800

1000Untreated uninfected

Treated uninfected

Untreated infected

Art 2x10mg/kg

1800

1500

1200*

*

200

400

600

300600900

1200 uninfectedUntreated* significant vs. Art .

*Chl 2x15mg/kg

Art & Chl

*Significant vs. Art treated

Conc

entr

atio

Conc

entr

atio

Conc

entr

atio

Conc

entr

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Conc

entr

atio

Conc

entr

atio

Conc

entr

atio

900

600

300 200

400

600

***

*

*Chl 2x15mg/kg

Art & Chl

*Significant vs. untreated infected

900

600

300

**

* * **

000 00


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FIG 8. Effect of artemisone and chloroquine on cytokines in P. bergheiANKA infection on day 12 post infectionANKA infection on day 12 post infection.Plasma cytokine levels in mice treated on days 6-8 (twice each day) post infection by artemisone (Art), chloroquine (Chl) or their combination. Columns and vertical bars represent mean ± SEM; n = 8-10/group. on June 29, 2018 by guest

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Table 1. A summary of drug effects on cytokine production in infected mice

Day post infection

Cytokine/Effects Artemisone Chloroquine Art+Chl

8 IL-4 = ↑ ↑ IL-10 ↑ ↑ ↑ IFN-γ = = ↓ TNF ↓ ↓ ↓

Positive effect 2 3 4 12 IL-4 = ↑ ↑

IL-10 ↑ = = IFN-γ ↑* ↑* = TNF ↓ ↓ ↓

Positive effect 1 1 2 ↑ (for IL-4 and IL-10) or ↓ (For IFN-γ and TNF) are effects related to alleviation of

CM ↑* (for IFN-γ) means effect that may increase CM = means no difference from control "Positive effect" is the cumulative number of effects that may alleviate CM, from

which the number of effects that may increase CM is reduced


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