Front. Microbiol. Frontiers in Microbiology Front. Microbiol. 1664-302X Frontiers Media S.A. 10.3389/fmicb.2017.00025 Microbiology Review A Systematic Review of In vitro and In vivo Activities of Anti-Toxoplasma Drugs and Compounds (2006–2016) Montazeri Mahbobeh 1 2 Sharif Mehdi 1 3 Sarvi Shahabeddin 1 3 Mehrzadi Saeed 4 Ahmadpour Ehsan 5 Daryani Ahmad 1 3 * 1Toxoplasmosis Research Center, Mazandaran University of Medical Sciences Sari, Iran 2Student Research Committee, Mazandaran University of Medical Sciences Sari, Iran 3Department of Parasitology and Mycology, Sari Medical School, Mazandaran University of Medical Sciences Sari, Iran 4Department of Pharmacology, School of Medicine, Iran University of Medical Sciences Tehran Iran 5Drug Applied Research Center, Tabriz University of Medical Sciences Tabriz, Iran

Edited by: Octavio Luiz Franco, Universidade Católica de Brasília, Brazil

Reviewed by: Osmar Nascimento Silva, Universidade Católica Dom Bosco, Brazil; Nasib Singh, Eternal University, India

*Correspondence: Ahmad Daryani daryanii@yahoo.com

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

 20 01 2017 2017 8 25 01 09 2016 05 01 2017 Copyright © 2017 Montazeri, Sharif, Sarvi, Mehrzadi, Ahmadpour and Daryani. 2017 Montazeri, Sharif, Sarvi, Mehrzadi, Ahmadpour and Daryani

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The currently available anti-Toxoplasma agents have serious limitations. This systematic review was performed to evaluate drugs and new compounds used for the treatment of toxoplasmosis. Data was systematically collected from published papers on the efficacy of drugs/compounds used against Toxoplasma gondii (T. gondii) globally during 2006–2016. The searched databases were PubMed, Google Scholar, Science Direct, ISI Web of Science, EBSCO, and Scopus. One hundred and eighteen papers were eligible for inclusion in this systematic review, which were both in vitro and in vivo studies. Within this review, 80 clinically available drugs and a large number of new compounds with more than 39 mechanisms of action were evaluated. Interestingly, many of the drugs/compounds evaluated against T. gondii act on the apicoplast. Therefore, the apicoplast represents as a potential drug target for new chemotherapy. Based on the current findings, 49 drugs/compounds demonstrated in vitro half-maximal inhibitory concentration (IC50) values of below 1 μM, but most of them were not evaluated further for in vivo effectiveness. However, the derivatives of the ciprofloxacin, endochin-like quinolones and 1-[4-(4-nitrophenoxy) phenyl] propane-1-one (NPPP) were significantly active against T. gondii tachyzoites both in vitro and in vivo. Thus, these compounds are promising candidates for future studies. Also, compound 32 (T. gondii calcium-dependent protein kinase 1 inhibitor), endochin-like quinolones, miltefosine, rolipram abolish, and guanabenz can be repurposed into an effective anti-parasitic with a unique ability to reduce brain tissue cysts (88.7, 88, 78, 74, and 69%, respectively). Additionally, no promising drugs are available for congenital toxoplasmosis. In conclusion, as current chemotherapy against toxoplasmosis is still not satisfactory, development of well-tolerated and safe specific immunoprophylaxis in relaxing the need of dependence on chemotherapeutics is a highly valuable goal for global disease control. However, with the increasing number of high-risk individuals, and absence of a proper vaccine, continued efforts are necessary for the development of novel treatment options against T. gondii. Some of the novel compounds reviewed here may represent good starting points for the discovery of effective new drugs. In further, bioinformatic and in silico studies are needed in order to identify new potential toxoplasmicidal drugs.

 Toxoplasma gondii toxoplasmosis drugs compounds in vitro in vivo Introduction

Toxoplasma gondii (T. gondii), an obligate intracellular, parasitic protozoan, is the etiologic agent of toxoplasmosis. About 30–50% of the world population is infected with the parasite, and it is the most prevalent infection among humans (Tenter et al., 2000; Flegr et al., 2014). Worldwide, over 1 billion people are estimated to be infected with T. gondii (Hoffmann et al., 2012). Its prevalence in some countries is high (e.g., Brazil, 77.5%; Sao Tome and Principe, 75.2%; Iran, 63.9%; Colombia, 63.5%; and Cuba, 61.8%) (Pappas et al., 2009). The annual incidence of congenital toxoplasmosis was estimated to be 190,100 cases globally (Torgerson and Mastroiacovo, 2013).

In the United States, the Centers for Disease Control and Prevention (CDC) reported that 22.5% of the population 12 years and older have been infected with Toxoplasma with 1.1 million new infections each year, making it the second most common cause of deaths due to foodborne diseases (an estimated 327 deaths) and the fourth leading cause of hospitalizations attributable to foodborne illness (an estimated 4428 hospitalizations). Also, an estimated 400–4000 infants are born with congenital toxoplasmosis in the United States each year (Jones et al., 2014).

T. gondii has three infectious stages of sporozoites (in oocysts), tachyzoites (rapidly multiplying form), and bradyzoites (tissue cyst form). Among them, tachyzoites are responsible for clinical manifestations and the acute phase of the disease. They are susceptible to the immune response of the host and to drug action. The resistant cyst form of the parasite is resistant to both the immune system and drugs (Hill and Dubey, 2002).

Acute toxoplasmosis in healthy individuals is usually subclinical and asymptomatic, but may lead to chronic infection. However, toxoplasmosis can lead to great morbidity and mortality rates in imunocompromised or congenitally infected individuals (Dubey and Jones, 2008; Ahmadpour et al., 2014). In AIDS patients, presence of the parasite causes necrotizing encephalitis and focal cerebral lesions in the central nervous system (CNS) from primary or recrudescent infection. In immunocompetent patients, latent toxoplasmosis occurs with the formation of cysts principally in the CNS (Martins-Duarte et al., 2006).

In the recent years, the development of well-tolerated and safe specific immunoprophylaxis against toxoplasmosis is a highly valuable goal for global disease control (Lim and Othman, 2014). Immunotherapeutics strategies for improving toxoplasmosis control could either be a vaccine which would induce strong protective immunity against toxoplasmosis, or passive immunization in cases of disease recrudescence. In the last few years, much progress has been made in vaccine research on DNA vaccination, protein vaccination, live attenuated vaccinations, and heterologous vaccination; while there were few candidates on passive immunization. New vaccine candidates have been tested, including in particular proteins from T. gondii ROP, MIC, and GRA organelles, multi-antigen vaccines, novel adjuvants but until now the researches could not access to a proper vaccine for prevention of toxoplasmosis in human (Zhang et al., 2013, 2015).

The recommended drugs for treatment or prophylaxis of toxoplasmosis are pyrimethamine and sulfadiazine. Unfortunately, these drugs have side effects such as neutropenia, severe drop of platelet count, thrombocytopenia, leucopenia, elevation in serum creatinine and serum liver enzymes, hematological abnormalities, and hypersensitivity reactions (Bosch-Driessen et al., 2002; Silveira et al., 2002; Schmidt et al., 2006). In addition, other drugs, such as azithromycin, clarithromycin, spiramycin, atovaquone, dapsone, and cotrimoxazole (trimethoprim-sulfamethoxazole), have been used for clinical toxoplasmosis. However, these drugs are poorly tolerated and have no effect on the bradyzoite form (Araujo and Remington, 1992; Petersen and Schmidt, 2003; Serranti et al., 2011).

In a clinical trial, 24% of sera positive women treated with spiramycin and pyrimethamine plus sulfadoxine combination delivered Toxoplasma infected infants in France (Bessières et al., 2009). Spiramycin monotherapy can be effective during the early stage of pregnancy to prevent prenatal transmission (Julliac et al., 2010). More than 50% of patients treated with spiramycin retained T. gondii DNA in blood and remained infected (Habib, 2008).

In recent years, studies have focused on finding safe drugs with novel mechanisms of action against T. gondii. Accordingly, there is an urgent need to evaluate new drugs based on novel and innovative therapeutic strategies against T. gondii that are both efficacious and nontoxic for humans (Rodriguez and Szajnman, 2012; Vanagas et al., 2012; Angel et al., 2013). Therefore, the goal of the present systematic review was to retrieve published studies related to in vitro and in vivo evaluation of drugs and compounds for the treatment of toxoplasmosis (2006–2016) in order to prepare comprehensive data for designing more accurate investigations in future.

 Methodology

This review followed the preferred reporting items for systematic reviews (PRISMA) guidelines (Moher et al., 2009).

Literature search, study selection, and data extraction

English databases, including PubMed, Science Direct, Scopus, Google Scholar, ISI Web of Science, and EBSCO, were systematically searched for papers on in vitro and in vivo evaluation of anti-Toxoplasma activities of drugs and compounds, published worldwide, from 2006 to 2016. The keywords included were: “Toxoplasmosis,” “T. gondii,” “Anti-Toxoplasma,” “Drug,” “Anticoccidial,” “Treatment,” “In vitro,” “In vivo,” and “Compound.”

Papers written in English were selected. Gray literature and abstracts of articles which were published in congresses were not explored. In addition, in order to avoid missing any articles, whole references of the papers were meticulously hand-searched. Among English articles found with the mentioned strategies, full text papers that used laboratory method both in vitro and in vivo were included.

Also, studies with at least one of the following criteria were excluded: (1) studies that were not relevant; (2) articles not available in English; (3) studies on treatments for ocular infection; (4) articles that were of review or descriptive study type; (5) articles which contained no eligible data; (6) case series reports; (7) the data were duplicated from other studies or we were unable to obtain them; (8) those that were on efficacy of anti-T. gondii medicines in humans; and (9) any drug with an IC50 value > 10 μM.

 Data collection

All the experimental studies that were carried out to evaluate the efficacy of either drugs or compounds against T. gondii both in vitro and in vivo were included, and replicates were excluded. The inclusion criteria for selection of in vitro studies were important information about medication used for the experiments, type of cells used for culture, identification of the Toxoplasma strain, laboratory methods used for assessing drug activities, and main results comprising of the 50% inhibitory concentration (IC50). We reported in vivo studies used animal models, Toxoplasma strain, route of infection, the treatment schedule (dosage, route of administration, duration of treatment), the criteria for assessing drug activity (mainly survival for acute toxoplasmosis, histology, and brain cyst burdens for chronic infection), and the main results.

 Results Analysis of the included literature

A total of 118 papers (83 studies in vitro, 59 in vivo, 27 both in vitro and in vivo) published from 2006 to 2016, were included in the systematic review. Figure 1 briefly shows the search process in this systematic review article.

The PRISMA flow diagram of the search strategy, study selection, and data management procedure of in vitro and in vivo activities of anti-Toxoplasma drugs and compounds (2006–2016).

 Mechanisms of action

In the current systematic review, 80 clinically available drugs (Table 1) and several new compounds with more than 39 pathways/ mechanisms of action were evaluated against T. gondii in both in vitro and in vivo studies. Several target based drug screens were also identified against T. gondii include mitochondrial electron transport chain, calcium-dependent protein kinase 1, type II fatty acid synthesis, DNA synthesis, DNA replication, etc. (Table 2). Also, drugs/compounds with known mechanisms of action on life stages of T. gondii are shown in Figure 2. Our collective data indicated that many of the drugs/ compounds evaluated against T. gondii act on the apicoplast. Therefore, the apicoplast represents as a potential drug target for new chemotherapy.

Clinically available drugs/compounds evaluated against T. gondii in vitro and in vivo studies.

Common clinical uses Drugs/compounds References
Antiprotozoal agents Bisphosphonates Baramee et al., 2006; Ferreira et al., 2006; Rajapakse et al., 2007; Strobl et al., 2007; Leepin et al., 2008; Shubar et al., 2008; Liesen et al., 2010; Aquino et al., 2011; Franco et al., 2011; Martins-Duarte et al., 2011; Chew et al., 2012; Asgari et al., 2013; Bilgin et al., 2013; Gomes et al., 2013; Gaafar et al., 2014; da Silva et al., 2015; El-Zawawy et al., 2015a,b
Diamidine analogs
Spiramycin (Rovamycin)
Thiosemicarbazides
4-thiazolidinones
1,3,4-thiadiazoles
Naphthalene-sulfonyl-indole
Thiosemicarbazone
Phenylsemicarbazone
Ivermectin
Silver nanoparticles
Novel ferrocenic atovaquone derivatives
Triclosan
Triclosan liposomal nanoparticles
Metronidazole
1,25(OH)2D3
Naphthoquinone
PHNQ6a
Novel azasterols
Apicidin
Antimalarial agents Pyrimethamine Meneceur et al., 2008; Mui et al., 2008; Doggett et al., 2012; Zhou et al., 2014; Jain et al., 2015
Atovaquone
Triazine JPC-2067-B
Spiroindolone
Endochin-like quinolones
Halofuginone
Antibacterial agents Sulfadiazine Meneceur et al., 2008; Costa et al., 2009; Barbosa et al., 2012; Payne et al., 2013; Castro-Filice et al., 2014; Gaafar et al., 2014; Martins-Duarte et al., 2015
Azithromycin
Enrofloxacin
Fusidic acid
Ciprofloxacin
Chitosan
Antiretroviral agents Atazanavir Monzote et al., 2013
Fosamprenavir
Indinavir
Nelfinavir
Ritonavir
Saquinavir
Anticoccidial agents NPPPb Kul et al., 2013; Choi et al., 2014; Oz, 2014a,b
Diclazuril
Toltrazuril
Antihelminthic agents Niclosamide Fomovska et al., 2012; Galván-Ramírez et al., 2013
Nitazoxanide
Antifungal agents Itraconazole Martins-Duarte Edos et al., 2008; Martins-Duarte et al., 2010, 2013; Gaafar et al., 2014
Fluconazole
Chitosan
Anticancer agents SAHAc Strobl et al., 2007; Portes Jde et al., 2012; Leyke et al., 2012; Barna et al., 2013; Kadri et al., 2014; de Lima et al., 2015; Eissa et al., 2015; Opsenica et al., 2015; Dittmar et al., 2016
Pterocarpanquinone
Ruthenium complexes
Quinoline derivatives 4-aminoquinoline
4-piperazinylquinoline analogs
Miltefosine
Tetraoxanes
Gefitinib
3-bromopyruvate
Tamoxifen
Immunosuppressants agents Auranofin Ghaffarifar et al., 2006; Wei et al., 2007; Andrade et al., 2014; Ihara and Nishikawa, 2014
Am80
Betamethasone
Pyridinylimidazole
Imidazopyrimidine
Immunomodulators agents Rolipram Afifi and Al-Rabia, 2015
Immunoregulatory agents Levamisole Köksal et al., 2016
Antipsychotic agents Aripiprazole Saraei et al., 2015
Antioxidant agents Resveratrol Bottari et al., 2015
Antischizophrenic agents Haloperidol Goodwin et al., 2011; Fond et al., 2014; Saraei et al., 2016
Clozapine
Fluphenazine
Trifluoperazine
Thioridazine
Amisulpride
Cyamemazine
Levomepromazine
Loxapine
Olanzapine
Risperidone
Tiapride
Moodstabilizing agents Valproate Fond et al., 2014
Anti hypertensive agents Guanabenz Benmerzouga et al., 2015
Anti hypertensive and irregular heart rate agents Propranolol Montazeri et al., 2015, 2016

2-hydroxy-3-(1′-propen-3-phenyl)-1,4-naphthoquinone.

(4-nitrophenoxy) phenyl] propane one.

Suberoylanilide hydroxamic acid.

Drugs/compounds with pathways/ mechanisms of action against T. gondii.

Pathway/mechanism of action Drugs/compounds References
Electron transport chain PHNQ6*a Baramee et al., 2006; Ferreira et al., 2006, 2012; Saleh et al., 2007; Meneceur et al., 2008; Bajohr et al., 2010; Doggett et al., 2012; Kul et al., 2013; de Lima et al., 2015
HDQ*b
Atovaquone*
Endochin-like quinolones*
Ferrocenic atovaquone derivatives
Naphthoquinones
Toltrazuril
3-Bromopyruvate
Sterol biosynthesis Novel quinuclidine (ER119884, E5700) Martins-Duarte et al., 2006
Synthesis of cholesterol Am80* Ihara and Nishikawa, 2014
Antifolate Pyrimethamine* Meneceur et al., 2008; Mui et al., 2008; Martins-Duarte et al., 2013
Sulfadiazine*
Dihydrotriazine*
(JPC-2067-B, JPC-2056)
Calcium-dependent protein kinase 1 1 NM-PP1* Sugi et al., 2011; Doggett et al., 2014; Moine et al., 2015b; Vidadala et al., 2016
Bumped Kinase Inhibitor 1294*
Imidazo [1,2-b] pyridazines*
Compound 32*
Human mitogen-activated protein kinase Pyridinylimidazole* Wei et al., 2007
Imidazopyrimidine*
Nucleoside triphosphate hydrolase (NTPase) 2-(Naphthalene-2-γlthiol)-1H indole* Asgari et al., 2013, 2015
Isoprenoid pathway 2- alkylaminoethyl- 1,1- bisphosphonic acids* Shubar et al., 2008; Szajnman et al., 2008; Li et al., 2013
Newly synthesized bisphosphonates*
Atorvastatin*
Type II fatty acid synthesis Thiolactomycin* Martins-Duarte et al., 2009; Tipparaju et al., 2010; El-Zawawy et al., 2015a,b
53 novel compounds*
Inhibitors of enoyl reductase
Triclosan and triclosan liposomal*
Protein synthesis Azithromycin* Costa et al., 2009; Franco et al., 2011; Chew et al., 2012; Zhou et al., 2014; Palencia et al., 2016
Spiramycin*
Spiroindolone
3-aminomethyl benzoxaborole (AN6426)
Disappearance of the Apicoplast Quinoline derivatives* Smith et al., 2007; Kadri et al., 2014
(MC1626, quinoline, 8-hydroquinoline and B23)
Histone deacetylase enzyme SAHA*c Strobl et al., 2007; Maubon et al., 2010; Kropf et al., 2012
SBHA*d
Scriptaid*
Trichostatin A*
Di-cationic pentamidine-analog*
FR235222, FR235222 derivative*
DNA synthesis Metronidazole* Liesen et al., 2010; Chew et al., 2012; Gomes et al., 2013
Phenylsemicarbazone*
Phenylthiosemicarbazones*
Thiosemicarbazides*
4-Thiazolidinones*
1,3,4-thiadiazoles*
Cyclic AMP signaling pathways Rolipram* Afifi et al., 2014; Afifi and Al-Rabia, 2015
Post-translational modification by N-linked glycosylation of proteins Tunicamycin* Luk et al., 2008
Membrane permeability Novel diamidine analog* Leepin et al., 2008
Microfilament functional Cromolyn sodium Endeshaw et al., 2010; Rezaei et al., 2014; Montazeri et al., 2015, 2016
Ketotifen
Propranolol
Oryzalin analogs
Micronemal secretion pathway, cysteine protease Peptidyl vinyl sulfone compounds* (LHVS and ZL3VS) Teo et al., 2007
Immuno-regulatory Levamisole* Köksal et al., 2016
Translational control Guanabenz* Payne et al., 2013; Benmerzouga et al., 2015; Jain et al., 2015
Fusidic acid
Halofuginone*
DNA gyrase activity, transcription Enrofloxacin Barbosa et al., 2012; Martins-Duarte et al., 2015
Ciprofloxacin derivatives*
Thioredoxin reductase Auranofin Andrade et al., 2014
Topoisomerases I and II HSP90 protein Harmane, norharmane, and harmine Alomar et al., 2013
Metabolism of neurotransmitters in the brain Resveratrol Bottari et al., 2015
Effect on the liver biochemical parameters ATT-5126 and KH-0562 Choi et al., 2014
Vascular ATP synthase subunit C and/or methyltransferase NPPP Choi et al., 2015
Sterol biosynthesis enzyme-sterol methyl transferase. 22, 26-azasterol and 24, 25-(R, S)- epiminolanosterol Martins-Duarte et al., 2011
Downregulates expression of serine/threonine protein phosphatase Diclazuril Oz, 2014a,b
Ergosterol synthesis Fluconazole Martins-Duarte Edos et al., 2008; Martins-Duarte et al., 2013
Itraconazole
Interruption of mitosis Trifluralin Wiengcharoen et al., 2007
Oxidative phosphorylation Niclosamide Fomovska et al., 2012
Apocynin-dependent pathway NSC3852 Strobl et al., 2009
Phospholipid metabolism Miltefosine Eissa et al., 2015
Quinone oxidoreductase expression Nitaxozanide Galván-Ramírez et al., 2013
Kinase inhibitors Small-molecules Kamau et al., 2012
Tyrosine kinase Gefitinib Yang et al., 2014
Crizotinib
Adenosine kinase in the purine salvage pathways N6-benzyladenosine analog* Kim et al., 2007; Szajnman et al., 2008
Purine nucleoside phosphorylase 3-(thiophen-2-yl)-1,2,4-triazole-5-thione Dzitko et al., 2014b
Damage on the microneme proteins 7-nitroquinoxalin-2-ones (VAM2-2) Fernández et al., 2016

Drugs/compounds with known pathway/mechanisms of action gainst T. gondii.

2-hydroxy-3-(1′-propen-3-phenyl)-1,4-naphthoquinone.

1-hydroxy-2-dodecyl-4 (1H) quinolone.

Suberoylanilide hydroxamic acid.

Suberic bishydroxamic acid.

Drugs/compounds with known mechanisms of action on life stages of T. gondii, tachyzoites (T), and bradyzoites (B). 1, apical end; 2, Cell membrane; 3, microneme; 4, cytosol; 5, endoplasmic reticulum; 6, core; 7, mitochondria; 8, apicoplast.

 The investigated strains

T. gondii has three main clonal lineages in population structure; type I (including a highly virulent RH strain), Type II (including ME49 and PRU, avirulent strains), and Type III (including avirulent strains like NED), which is correlated with virulence expression in mice (Howe and Sibley, 1995).

In vitro and in vivo screening methods were used of type I T. gondii (mostly RH strain; 76 studies in vitro, and 36 in vivo). Because type I RH strain is highly virulent in mice, causing 100% mortality, but types II and III are relatively less virulent. Although in some studies, ME49 (7 studies in vitro, and 17 in vivo), Prugniaud, EGS, and VEG strains were used, which showed that the outcome of infections depends on the challenge dose and on the genotype of the host (Szabo and Finney, 2016). Details about the investigated strains in vitro and in vivo are shown in Tables 3, 4, respectively.

Summary of in vitro studies evaluated the anti-Toxoplasma activity of drugs/compounds.

No Drug Strain Cells Culture Evaluation Main results Effectivity Positive control References
1 Two novel quinuclidine (ER119884, E5700) RH LLCMK2 24, 48 h IC50 valuesa IC50 ER119884, E5700 = 0.66, 0.23 μM Effective Sulfadiazine, pyrimethamine Martins-Duarte et al., 2006
2 Fourteen novel ferrocenic atovaquone derivatives 76K, PLK, A to R HFF 48 h IC50 values IC50 2d, 2e, 2f = 5.0, 2.5, 6.25 μM Effective 2d, 2e, 2f – Baramee et al., 2006
3 Betamethasone and IFN-γb RH Hela 24, 48, 72 h Counting the number of tachyzoites High number of plaques was seen in group with 40 μg/ml of betamethasone. Betamethasone not effective, IFN–γ effective – Ghaffarifar et al., 2006
4 Suberoylanilide hydroxamic, suberic bishydroxamic acid, scriptaid, trichostatin A RH HS68 HFF 48, 72 h IC50 values IC50 scriptaid = 0.039 μM Scriptaid was the most effective – Strobl et al., 2007
5 RWJ67657, RWJ64809c, RWJ68198d RH, ME49 HFF 48 h IC50 values RWJ67657 was at least as potent as RWJ68198, SB203580, or SB202190 in reducing of T. gondii replication RWJ67657, SB203580 effective – Wei et al., 2007
6 Novel drug compounds (A–I) (B,F,G,H) (trifluralin analogs) RH Vero 72 h MTT assaye, crystal violet assay IC50 drug F = 10 μM Drugs F was the most effective – Wiengcharoen et al., 2007
7 1-hydroxy-2-dodecyl-4(1H) quinolone (HDQ) RH HFF 24 h Replication rate determined IC50 HDQ = 0.0024 ± 0.0003 μM Effective – Saleh et al., 2007
8 Quinoline derivative MC1626 RH HFF 24 h Standard [3H]uracil uptake and plaque assays 100 μM reducing growth Effective – Smith et al., 2007
9 N6-benzyladenosine analogs RH HFF 24 h MTT assay IC50 N6-(2,4-dimethoxybenzyl) Adenosine = 8.7 ± 0.6 μM, exhibited the most favorable activity Effective Sulfadiazine, pyrimethamine Kim et al., 2007
10 Fluorine-containing aryloxyethyl thiocyanate derivatives RH HFF 24 h IC50 values IC50 compounds 1 and 3 = 2.80 and 3.99 μM Effective Atovaquone Liñares et al., 2007
11 LHVS, ZL3VSf RH or 2F1 HFF 45 min B galg, Red/green invasion assay, SDS-PAGE, immunoblotting, gliding motility assay IC50 LHVS and ZL3 VS = 10 and 12.5 μM Effective 3,4-dichloroisocoumarin Teo et al., 2007
12 1,25(OH) 2D3 RH MICc12 72 h Trypan blue assay Ruled out any toxic effects of 1,25(OH) 2D 3 for T. gondii Effective – Rajapakse et al., 2007
13 Tunicamycin RH HFF 2, 24, or 48 h Fluorescence and electron microscopy N-Glycosylation is completely inhibited by treatment of parasites with tunicamycin Effective Pyrimethamine Luk et al., 2008
14 Novel diamidine analogs RH Vero HFF 2 or 3 days IC50 values, Q-PCRh IC50 DB750, DB786 = 0.16, 0.22 μM Effective – Leepin et al., 2008
15 Pyrimethamine, sulfadiazine, and atovaquone 17 strains T. gondii THP-1 MRC-5 7 days IC50, real-time PCR IC50 pyrimethamine = 0.0002, 0.01 μM Effective – Meneceur et al., 2008
IC50 atovaquone = 0.0001, 0.00005 μM
IC50 sulfadiazine = 0.01, 0.07 μM for
13 strains and were > 0.1 μM for three strains
16 Novel triazine JPC-2067-B RH HFF 3 days Liquid scintillation counting IC50 JPC-2067-B = 0.02 μM, Effective – Mui et al., 2008
IC90 JPC-2067-B = 0.05 μM
17 Newly synthesized bisphosphonates (15 new compounds) RH Mouse macrophages (J 744A.1) 24, 48 h MTT assay, flow cytometry 91A and 282A showed moderate and low toxicity (cell viability between 70% and 100%) Effective – Shubar et al., 2008
18 2-alkylaminoethyl- 1,1-bisphosphonic acids RH HFF Daily IC50 values, radiometric assay IC50 compound 19 = 2.6 μM Compound 19 was very effective . Szajnman et al., 2008
19 Itraconazole RH LLCMK2 24 or 48 h IC50 values, TEMi analysis IC50 = 0.11, 0.05 μM for 24, 48 h Effective – Martins-Duarte Edos et al., 2008
20 Thiolactomycin analogs (8 new compounds) RH LLCMK2 24, 48 h IC50 values, Lipid extraction, chromatographic analysis IC50 compounds = 1.6-29.4 μM Compound 5 was very effective Sulfadiazine, pyrimethamine Martins-Duarte et al., 2009
21 NSC3852j RH HS 68 HFF 2 h SYBR green assay, MTS assay, ROS assay, NO assays EC50 NSC3852 = 0.08 μM, NSC3852, NSC74949 were the most effective – Strobl et al., 2009
EC50 NSC74949 = 0.6 μM
22 FR235222, FR235222 derivative compounds (W363, W371, W399, W406, W425) RH, PRU (type II) HFF 24 h EC50 determination, Western blot analysis, immunofluorescence microscopy 100% altered cysts 24 h after treatment with the lowest concentration of FR235222 Effective – Maubon et al., 2010
23 Thiosemicarbazides, 4-thiazolidinones and 1,3,4-thiadiazoles RH Vero 24 h Mean number of intracellular parasitesa, LD50k A significant decrease in the percentage of infected cells and in the mean number of tachyzoites per cell from the concentrations of 0.1, 1, 10 mM Effective Hydroxyurea, sulfadiazine Liesen et al., 2010
24 FLZl and ITZm RH LLCMK2 24, 48 h IC50 values IC50 FLZ = 8.9, 3.1 μM after 24, 48 h Effective Sulfadiazine, pyrimethamine Martins-Duarte et al., 2010
IC50 ITZ = 0.1, 0.05 μM for 24, 48 h
25 1-Hydroxy-2-Alkyl-4(1H) Quinolone Derivatives RH (type I) HFF 24 h IC50 values IC50 compound A, B = 0.0004, 0.0008 μM Effective Atovaquone Bajohr et al., 2010
26 Oryzalin Analogs RH HFF 8 day 26 h Plaque assay, Immunofluorescence assay, IC50 values IC50 18b = 0.03 μM Effective – Endeshaw et al., 2010
27 53 novel compounds (Inhibitors of Enoyl reductase) RH HFF 3 days IC50 values IC50 compounds 2, 19 = 0.04, 0.02 μM, Compounds 2, 19, 39 greatest effect – Tipparaju et al., 2010
IC50 compounds 39 less active
28 Haloperidol, clozapine, fluphenazine, trifluoperazine, thioridazine RH HFF 48 h IC50 values IC50 fluphenazine, thioridazine, trifluoperazine = 1, 1.2, and 3.8 μM Fluphenazine, thioridazine, trifluoperazine were effective – Goodwin et al., 2011
29 Azithromycin, spiramycin RH Bewo cell line 24 h MTT assay, measurement of Th1/Th2 Increase TNF-an, IL-10, IL-4 production, but decreased IFN-γ Effective – Franco et al., 2011
30 Novel azasterols RH ME49 LLCMK2 24 or 48 h IC50 values, imunofluorescence assays IC50 compounds 1, 2, 3 = 0.8–4.7 μM Compound 3 was the most effective – Martins-Duarte et al., 2011
31 Ciprofloxacin derivatives RH LLC-MK2 24 or 48 h IC50, MTS assay IC50 compounds 2, 4, 5= 0.42, 1.24, and 0.46 μM Effective – Dubar et al., 2011
32 2-hydrazolyl-3-phenyl-5-(4-nitrobenzylidene)-4-thiazolidinone substituted RH Vero 24 h LD50 values LD50 = 0.5, 10 mM Effective Hydroxyurea, Sulfadiazine Aquino et al., 2011
33 Nanoparticles RH (CAT-GFP) Macrophages J 774-A1 3 day HPLCo: flow cytometry Cap.85% observed maximum in Toxoplasmosis therapy efficiency Effective – Leyke et al., 2012
34 Enrofloxacin RH HFF 72 h MTT assays Enrofloxacin resulted in a significant inhibition of the percentage of infected cells by the parasite (58.72%) Effective Sulfadiazine, pyrimethamine Barbosa et al., 2012
35 ELQ-271 and ELQ-316q 2F HFF 4 days Host-cell toxicity IC50 ELQ-271, ELQ-316 = 0.0001, and 0.000007 μM Effective Atovaquone Doggett et al., 2012
36 Pterocarpanquinone RH LLCMK2 24 or 48 h Direct counts, viability, imunofluorescence assays IC50= 2.5 μM Effective – Portes Jde et al., 2012
37 New naphthoquinones and an alkaloid RH, EGS HFF 48 h MTT assays IC50 QUI-5, and QUI-6r = 69.35, and 172.81 μM Effective Atovaquone, Sulfadiazine Ferreira et al., 2012
38 Spiramycin coadministered with metronidazole ME49 Vero E6 1 week Numbers of cysts and tachyzoites Spiramycin reduced in vitro reactivation, metronidazole alone did not have significant effect Effective – Chew et al., 2012
39 Di-cationic pentamidine-analogs RH ME49 HFF 72 h Cytotoxicity assays IC50 arylimidamide DB745 = 0.11, 0.13 μM (tachyzoites of Rh, Me49) Effective Atovaquone Kropf et al., 2012
40 Small-Molecule (n=527) Strains 5A10 (type III strain) HFF 72 h Luciferasebased assay, Host cell viability, electron microscopy, invasion, motility assays EC50 s for the 14 compounds = 0.14–8.7 μM 14 compounds effect – Kamau et al., 2012
41 Salicylic acids (39 compounds) RH, RH-YFP, and ME49 HFF 1 h [3H]-Uracil incorporation and YFP Fluorescence assay 3i, 3j, 7a, 14a, and 14b were active at low nanomolar concentrations Effective Pyrimethamine, Sulfadiazine Fomovska et al., 2012
42 FLZ combined with sulfadiazine and pyrimethamine RH LLCMK2 24 h IC50 values and MTS assay IC50 FLZ = 8.4 ± 1.2, IC50 sulfadiazine/pyrimethamine, pyrimethamine = 8.7 ± 0.8 μM Effective – Martins-Duarte et al., 2013
43 Harmane, Norharmane (β-carboline alkaloids) RH Vero HFF 1, 24 h Parasite invasion and replication rate harmane and harmine showed 2.5- to 3.5-fold decrease in the invasion rates at doses of 40 μM, norharmane 2.5 μM Effective Sulfadiazine Alomar et al., 2013
44 Fusidic acid Prugniaud HFF 7 days Lytic plaques counted IC50 = 7.7 μM, decreased the number of T. gondii plaques in a dosedependent manner Effective – Payne et al., 2013
45 Two naphthalene-sulfonyl-indole compounds RH – 1.5 h Stained by PI, analyzed by FACS LD50 compound A, B = 62, 800 μmol Effective Saponin Asgari et al., 2013
46 (Benzaldehyde)-4-phenyl-3- thiosemicarbazone, (benzaldehyde)-(4 or 1)- phenylsemicarbazone (9 compounds) RH Vero 24 h Cytotoxicity, number of intracellular parasites LD50 compound 8 = 0.3 mM, reduced the number of intracellular parasites by 82 % in a concentration of 0.01 mM Effective Sulfadizine Gomes et al., 2013
47 Ivermectin and sulphadiazine RH Hep- 2 24, 48, 72 h IC50, invert microscopy, ELISA assay IC50 ivermectin and sulphadiazine = 0.2, and 29.1 μM Effective – Bilgin et al., 2013
48 Novel ruthenium complexes RH HFF 72 h cytotoxicity assessment, TEM EC50 compounds 16, 18 = 18.7, 41.1 nM Compounds 16, and 18 effective – Barna et al., 2013
49 Atazanavir, fosamprenavir, indinavir, nelfinavir, ritonavir, and saquinavir RH Macrophages Swiss Webster 48 h IC50 determination, MTT assay IC50 atazanavir ritonavir, and saquinavir = > 1 μM Effective Pyrimethamine Monzote et al., 2013
IC50 fosamprenavir, and nelfinavir = > 5 μM
50 Atorvastatin RH HFF 8 days IC50 values IC50 = 50 μM Effective – Li et al., 2013
51 Nitaxozanide RH Astrocyte 24,48 h Immunocytochemical method, microscopic analysis, viability Nitazoxanide produced 97% T. gondii death in a concentration of 10 mg/mL in 48 h infected astrocytes Effective Pyrimethamine Galván-Ramírez et al., 2013
52 Amisulpride, cyamemazine, fluphenazine, haloperidol, levomepromazine, loxapine, olanzapine, risperidone, tiapride, and valproate RH HFF 4 h Growth inhibition assay Amisulpride, tiapride and valproate did not have inhibitory activity Zuclopenthixol, high effective – Fond et al., 2014
53 Spiroindolone RH HFF 72 h Fluorescence assays, cytotoxicity assessment IC50 = 1 μM Effective Pyrimethamine, sulfadiazine Zhou et al., 2014
54 Auranofin RH HFF 5 days Invasion and replication assays and plaque assays TD50 = 8.21 μM, IC50 = 0.28 μM Effective Pyrimethamine, Sulfadiazine Andrade et al., 2014
55 Azithromycin 2 F1 Placental tissues 48 h Production of cytokines and hormones Increases IL-6 production, reduced secretion of estradiol, progesterone, and HCG + β Effective Pyrimethamine, Sulfadiazine, folinic acid Castro-Filice et al., 2014
56 6-Trifluoromethyl-2-thiouracil (ATT-5126), (KH-0562) RH Hela 24 h MTS assay, IC50 IC50 ATT-5126, KH-0562 = 19.7, 32.2 μM Effective Pyrimethamine Choi et al., 2014
CC50 ATT-5126, KH-0562 = 35.4, 56.3 μM
57 Cromolyn sodium and ketotifen RH Macrophage monolayer 24 h Inhibition rate After 60 min the best efficacy was observed at 15 μg/ml (78.9 ± 1.70, 91.97 ± 0.37%) Effective – Rezaei et al., 2014
58 200 drug-like and 200 probe-like compounds of Malaria Box TS-4 (mutant of the RH) HFF 24 h Cytotoxicity assays Seven compounds with IC50 < 5 μM, SI > 6 7 compounds effected Pyrimethamine, sulfadiazine Boyom et al., 2014
59 Am80 RH, PLK, its recombinants J 774A.1 20 h Uracil incorporation assay, RT–PCRs, flow cytometry Am80 inhibited parasite growth by decreasing intracellular accumulation of cholesterol Effective – Ihara and Nishikawa, 2014
60 Pyrimethamine –loaded lipid-core nanocapsules RH LLC-MK2 72 h MTS assay TC50 PYR loaded lipid-core nanocapsules = 6.0 μM Effective – Pissinate et al., 2014
61 Quinoline derivatives (58 compounds) 2F HFF 4 days Cytotoxicity assays IC50 B23 = 0.4 ± 0.03 μM, the most effective compound 32 compounds effected – Kadri et al., 2014
62 74 novel thiazolidin-4-one derivatives – HFF 5 days Cytotoxicity assays IC50 derivatives 12 A, 27 A = 0.9, 2.9 μM Effective Timethoprim D'Ascenzio et al., 2014
63 Gefitinib and Crizotinib RH Hela 24, 48, 72 h Counting the number of T. gondii per parasitophorous vacuolar membrane Gefitinib inhibited the growth of T. gondii over 5 μM whereas Sunitinib did not Gefitinib effected Pyrimethamine Yang et al., 2014
64 1,4-disubstituted thiosemicarbazides RH Mouse L929 fibroblasts 24 h MTT assay and q-pcr 1g, 2b, 3d, 3l showed significant anti-parasitic effects 1 g was very effective Sulfadiazine Dzitko et al., 2014a
65 3-(thiophen-2-yl)-1,2,4-triazole-5-thione RH Mouse L929 fibroblasts 24 h IC50 values and q-pcr IC50 at least 30 times better than that of sulfadiazine Effective Sulfadiazine Dzitko et al., 2014b
66 1-[4-(4-nitrophenoxy) phenyl]propane-1-one (NPPP) RH Hela 24 h CC50, EC50 values EC50, CC50 = 36.2 ± 0.2, 67.0 ± 0.2 μM Effective – Choi et al., 2015
67 C-type lectin from Bothropspauloensis venom RH Hela 24 h MTT assay, cytokine measurements MTT assay between 0.195, 12.5 μg/mL MIF, IL-6 productions were increased Effective – Castanheira et al., 2015
68 Ciprofloxacin derivatives Compounds (2, 4, 5) RH LLCMK2 HFF 24, 48, 72 h Immunofluorescence, TEM Inhibited parasite replication early in the first cycle of infection Effective – Martins-Duarte et al., 2015
6 h
69 New chiral N-cylsulfonamide bis-oxazolidin-2-ones RH MRC-5 – IC50 values IC50 of Mol 1 was less than Mol 2 Effective Sulfadiazine Meriem et al., 2015
70 Guanabenz ME49 Prugniaud HFF 32 h EC50 values EC50 = 6 μM Effective – Benmerzouga et al., 2015
71 3-Bromopyruvate, Atovaquone RH LLC-MK2 24, 48 h, or 6 days Light-microscopic analysis, indirect immunofluorescent assays 73 and 71% reduction in intracellular parasites after 24, 48 h Effective – de Lima et al., 2015
72 Biphenylimidazoazines RH HFF 96 h EC50 values and fluorescence microscopy assay EC50 < 1 μM Effective Pyrimethamine Moine et al., 2015a
73 Halofuginone RH HFF 24 h EC50 values EC50 = 0.94 nM Effective Pyrimethamine Jain et al., 2015
74 Naphthoquinone derivative RH LLC-MK2 24, 48 h IC50 and MTT assay IC50 LQB 151 = < 1 μM Effective – da Silva et al., 2015
75 Aryloxyethyl thiocyanates RH Vero 24 h Determination of ED50 ED50 derivatives 15 and 16 = 1.6 μM and 1.9 μM Effective – Chao et al., 2015
76 Imidazo [1,2-b] pyridazines derivatives RH-GFP HFF 24 h Cytotoxicity assay EC50 16a, 16f = 100, 70 nM Effective – Moine et al., 2015b
77 Nitrofurantoin RH Hela 24 h MTS assay Selectivity = 2.3 Effective Pyrimethamine Yeo et al., 2016
EC50 = 14.7 μM
78 Quinoxalinone derivatives RH HEp-2t 24 h IC50 values, viability, invasion, and intracellular growth MIC50 VAM2-2 = 3.3 ± 1.8 μM VAM2-2 was very effective – Fernández et al., 2016
79 1120 compounds RH-GFP HFF 72 h Parasite invasion, Microneme secretion, Luciferase, and LC3-GFP assays 94 compounds with IC50 < 5 μM Tamoxifen effective – Dittmar et al., 2016
80 3-aminomethyl benzoxaborole (AN6426) RH HFF 24 h Determination of EC50 EC50 = 76.9 μM Effective Pyrimethamine Palencia et al., 2016
81 Sulfur-containing linear bisphosphonates RH, Prugniard Human fibroblasts (hTert cells) 5 days Determination of EC50 EC50 = 0.11 ± 0.02 μM Compound 22 was very effective – Szajnman et al., 2016
82 Fluorine-containing Analogs of WC-9 (4-phenoxyphenoxyethyl thiocyanate) RH Vero 24 h Determination of EC50 EC50 3-(3-fluorophenoxy), 3-(4-fluorophenoxy) phenoxyethyl thiocyanates, and 2-[3-(phenoxy)phenoxyethylthio]ethyl-1,1-bisphosphonat = 1.6 4.9 and 0.7 μM Effective – Chao et al., 2016
83 6-(1,2,6,7-tetraoxaspiro[7.11] nonadec-4-yl)hexan-1-ol (N-251) RH Human hepatocyte, Huh-7 72 h IC50 values, q-pcr, ultrastructural Change by TEM LC50 = 1.11 μg/ml Effective Sulfadiazine Xin et al., 2016

Half maximal inhibitory concentration.

Interferon gamma.

Pyridinylimidazole.

Imidazopyrimidine.

3− (4, 5−Dimethyl−2−Thiazyl) − 2, 5−Diphenyl−2H−Tetrazoliu Bromide.

Morpholinourea-leucyl-homophenolalaninyl-phenyl-vinylsulfone, N-benzoxycarbonyl-(leucyl) 3-phenyl-vinyl-sulfone.

B galactosidase.

Quantitative polymerase chain reaction.

Transmission electron microscopy.

5- nitroso-8-quinolinol.

Lethal Dose, 50%.

Fluconazole.

Itraconazole.

Tumor necrosis factor.

High Performance Liquid Chromatography.

Carriers achieved.

Endochin-like quinolones.

7-(4-methyl-3-pentenyl)-2-pyrrolidine-[1, 4]-naphthoquinone (QUI-5), 6-(4-methyl-3-pentenyl)-2-pyrrolidine-[1, 4]-naphthoquinone (QUI-6).

Reverse transcription polymerase chain reaction.

Human larynx epidermoid carcinoma epithelial cells.

Summary of in vivo studies evaluated the anti-Toxoplasma activity of drugs/ compounds.

No Drug Animal Strain Type of infection Inoculum Treatment Assessment of efficacy Main results Effectivity Positive control References
1 PHNQ6a alone or combined with sulfadiazine Female Swiss mice RH EGS P Acute, chronic 1000 tachyzoites (ip) 10 brain cysts (orally) PHNQ6 50 mg/kg/day Sulfadiazine, 40 mg/L Survival rates, IFATb, and liver histology Treatment protected at least 70, 90% of mice infected with RH and EGS strains Effective Sulfadiazine Ferreira et al., 2006
2 1, 25(OH) 2D3 BALB/c ME49 Acute 20 cysts 0.5 μg/kg/2 days ip Histopathology, RT-PCRc Low parasitic burdens were found Effective – Rajapakse et al., 2007
3 Pyridinylimidazole (RWJ67657, RWJ64809), imidazopyrimidine (RWJ68198) Female CBA/J, CD8 / – RH ME49 Acute 1000, 100, and 20 tachyzoites 3.8, 7.5, 15, 30, or 60 mg/kg i.p Survival rates The highest dose (60 mg/kg) significantly improved survival RWJ67657 effective – Wei et al., 2007
4 Novel triazine JPC-2067-B Outbred Swiss Webster RH Acute 10000 tachyzoites i.p 1.25 mg/kg/day orally Peritoneal T. gondii burden Intraperitoneal parasite numbers were reduced Effective – Mui et al., 2008
5 Newly synthesized bisphosphonates NMRI RH Acute 100 000 tachyzoites i.p 490, 1000, 512, 44.05, and 47.6 μM Flow cytometry Therapeutic efficacy was 100% for bisphosphonates 2F, 3B, 18A, 22A, and 30B Effective – Shubar et al., 2008
6 Azithromycin, Artemisia annua, spiramycin, SPFA Females C. callosus ME49 Chronic 20 cysts Azithromycin (9 mg/24 h), A. annua (1.0 mg/8 h), spiramycin (0.15 mg/8 h) Morphological, immunohistochemical analyses, mouse bioassay, and PCRd No morphological changes were seen in the placenta and embryonic tissues from females treated with azithromycin, spiramycin, and SPFA Azithromycin more effective – Costa et al., 2009
7 Dihydroartemisinin and azithromycin Kunming mice . Acute 2 × 103tachyzoites Dihydroartemisinin and azithromycin 75 and 200 mg/kg The ultrastructure of tachyzoites The ultrastructure of tachyzoites was observed in the treatment groups such as edema, enlarged, broken or damaged Effective – Yin et al., 2009
8 FLZf and ITZg Outbred female Swiss CF1 ME49 Chronic 20 cysts of the ME49 orally or i.p 10,20 mg/kg/day orally Survival rates and brain cyst burden ITZ survival of 90, 87% FLZ survival rate of 71, 85% Effective Sulfadiazine, pyrimethamine Martins-Duarte et al., 2010
9 HDQh derivatives Female NMRI, IRF-8 /– RH ME49 Acute, chronic 105 green fluorescent protein, i.p 10 cysts 32 mg/kg body weight/day Parasite loads in lungs, livers by qPCRe, and flow cytometry analyses Derivatives of HDQ had lower parasite concentrations than mice treated with HDQ Effective Atovaquone Bajohr et al., 2010
10 FR235222, FR235222 derivative, (W363, W371, W399, W406, W425) Outbred female Swiss PRU Chronic Living cysts i.p 200 nM Presence or absence of cysts in brain was assessed by staining No cysts were detected in mice inoculated with FR235222-treated Effective Pyrimethamine Maubon et al., 2010
11 Azithromycin combined with metronidazole BALB/c – Acutly 50 tissue cysts orally or i.p 250, 200 mg/kg/day Microscopical examination, bioassay were done for brain, and survival rates Cure rate 100% Effective – H.Al-jader and Al-Mukhtar, 2010
12 Novel compounds 2,19 (Inhibitors of Enoyl Reductase) CD1 RH Acute 2000 10 mg/kg i.p Parasite burdens in the peritoneal cavity and survival rates Reduction of parasite burden Effective – Tipparaju et al., 2010
13 SDS-coated atovaquone C57BL/6 ME49 Acute, chronic 10 cysts orally 100 mg/kg Histology, PCR Parasite loads and inflammatory changes in brains were significantly reduced Effective – Shubar et al., 2011
14 1NM-PP1 Old female ICR strain RH Acute 1.0 × 105 tachyzoites i.p 5 μM orally Survival rates, parasite load by qPCR Reduced the parasite load in the brains, livers, lungs Effective – Sugi et al., 2011
15 Enrofloxacin Calomys callosus, C57BL/6 RH ME49 Acute, chronic 100 tachyzoites RH strain 20 cysts per 100/ l (orally) Subcutaneously for 3 days, 3 mg/kg twice a week for the duration of 25-day Histological analysis, immunohistochemical assay, survival, cyst counts diminished significantly the tissue parasitism as well as the inflammatory alterations in the brain Effective Sulfadiazine, pyrimethamine Barbosa et al., 2012
16 Small-Molecule (C1, C2, C3, C5) BALB/c 5A10, PB3-10 Acute, chronic 10,000 tachyzoites i.p 4.4 mg/kg/day Survival rates, recording the total number of photons per second from each mouse C2 showed a significant reduction in parasite load in acute and reduced levels of parasite proliferation and increased survival in chronic phase C2 effective – Kamau et al., 2012
17 Endochin-like quinolones (ELQ-271, ELQ-316) Female CF-1 CBA/J RH ME49 Acute, chronic 20000 tachyzoites (express YFP) i.p 18 cysts of ME49 50, 20, 5, 1 mg/kg for 5 day Counted by flow cytometry ED50 values of 0.14, 0.08 mg/kg reducing cyst burden by 76–88% Effective Atovaquone Doggett et al., 2012
5 or 25 mg/kg for 16 day
18 Spiramycin coadministered with metronidazole Male BALB/c ME49 Chronic 1000 tachyzoites orally 400 mg/kg daily for 7 days Brain cysts counted Metronidazole increased spiramycin brain penetration, causing a significant reduction of T. gondii brain cysts Metronidazole alone showed no effect – Chew et al., 2012
500 mg/kg daily
19 New naphthoquinones, an alkaloid Female Swiss-Webster EGS Chronic 10 tissue cysts orally 50 μg/mL of QUI-11, 100 μg/mL of either QUI-6 or QUI-11 Presence of tachyzoites in the peritoneal cavities and survival rates The survival rates increased Effective Atovaquone Ferreira et al., 2012
20 Prednisolone Swiss albino RH ME49 Acute, chronic 1 × 104 tachyzoites, iP 235, 470, 705 mg/kg Number of tachyzoites present Greatly improved the number of tachyzoite, cyst forms in mice No effective – Puvanesuaran et al., 2012
21 Salicylic acids compounds 14a, 14b Swiss Webster RH, RH-YFP, ME49 Acute Oocysts orall gavage 100 or 25 mg/kg orally Survival rates Increased survival by 1 day Effective Pyrimethamine, sulfadiazine Fomovska et al., 2012
22 Atorvastatin Female Swiss Webster, BALB/c RH TATi Acute 5–20 tachyzoites i.p 10,000–100,000 tachyzoites 20 mg/kg/day ip Plaque assays and containing tachyzoites in peritoneal fluid Atorvastatin protect mice against death, cures a lethal infection Effective – Li et al., 2013
23 Fusidic acid Female BALB/c Prugniaud Acute 5 × 103 or 5 × 104 tachyzoites i.p 20 mg/kg Parasite burdens, analyses of host cytokine, and survival rates There was no statistically significant difference between mice treated with fusidic acid versus saline No effective Trimethoprim, sulfadiazine Payne et al., 2013
24 FLZ combined with sulfadiazine, and pyrimethamine CF1 RH Acute 103 tachyzoites 10 mg/kg/day of fluconazole with 40/1 mg/kg/day sulfadiazine, pyrimethamine Survival rates 93% survival Effective Sulfadiazine, pyrimethamine Martins-Duarte et al., 2013
25 Two naphthalene-sulfonyl-indole compounds BALB/c RH Acute 2 × 106 tachyzoites 25–800 μmol i.p Survival rates, liver touch smears with giemsa stained Both of the compounds was preserved Effective Asgari et al., 2013
26 Toltrazuril lambs ME49 Chronic 1 × 105oocysts 20, 40 mg/kg orally 2 times, once every week Presence of tissue cysts by histopathology, immunohistochemistry, and nested-PCR Cyst presence was determined as 44.4% Effective Kul et al., 2013
27 Auranofin Chicken embryos RH Acute 1 × 104 tachyzoites chorioallantoic vein 1 mg/kg Histopathology, immunohistochemistry, and qPCR Significantly reduced parasite load Effective Pyrimethamine, sulfadiazine Andrade et al., 2014
28 Spiroindolone Mice RH Acute 2000 tachyzoites 100 mg/kg/day Parasite burdens, measuring the fluorescence intensity Reduced the parasite burden in mice by 90% Effective – Zhou et al., 2014
29 6-Trifluoromethyl-2-thiouracil KH-0562i and ATT-5126j ICR female RH Acute 1 × 105 tachyzoites 100 mg/kg KH-0562i or ATT-5126j orally Measuring amount of the tachyzoites in mice ascites,LPOk, GSHl, ALTm, ASTn in mouse liver LPO level-KH-0562 and ATT-5126 = 87.4 and 105.2 nmol/g KH-0562 more effective Pyrimethamine Choi et al., 2014
30 Pyrazolopyrimidine-1294 BALB/c RH Pru Acute, chronic 105 tachyzoites 100, 30 mg/kg/day for 5 days Survival rates and number of T. gondii per ml Decreasing the numbers of T. gondii tachyzoites at both 100, 30 mg/kg Effective – Doggett et al., 2014
31 6-Trifluoromethyl-2-thiouracil KH-0562i, ATT-5126j Female ICR RH Acute 1 × 105 tachyzoites 100 mg/kg Proteomic profiles of T. gondii tachyzoites Decreased the amount of tachyzoites, mean numbers of tachyzoites = (66.8 ± 0.8) × 106 Effective Pyrimethamine Choi et al., 2014
32 Cromolyn sodium, ketotifen Balb/c RH Acute 4 × 105 tachyzoites Ketotifen 1, 2 mg/kg, cromolyn sodium 5, 10 mg/kg, ip Inhibition evaluated under a light microscope with giemsa staining After 60 min ketotifen at 2 mg/kg (69.83 ± 2.25 %), cromolyn sodium, at 10 mg/kg in (80.47 ± 2/49 %) had the best effect Effective – Rezaei et al., 2014
33 Diclazuril plus atovaquone CD1 mice PTG Strain Chronic 600 tachyzoites-i.p 65, 120 mg/kg diclazuril Hematoxylin eosin,Giemsa,immuno histochemical staining Combination diclazuril plus atovaquone was safe Effective – Oz, 2014b
34 Diclazuril plus atovaquone CD1 mice PTG strain Chronic 300, or 600 tachyzoites i.p 65, 120 mg/kg diclazuril Hematoxylin and eosin, slides evaluated of colonic tissues Combined therapy synergistically normalized pathology and to a lesser degree monotherapy Effective – Oz, 2014a
35 Am80 BALB/c mice RH, PLK Acute 1 × 10 3 tachyzoites i.p 1 mg/kg Survival rates Percent survival of mice increased statistically Effective – Ihara and Nishikawa, 2014
36 Chitosan and silver nanoparticles Swiss albino RH Acute 3.5 × 103 tachyzoites i.P 100, 200 μg/ml Parasite density and ultrastructural parasite changes Statistically significant decrease in the mean number of the parasite count in the liver and the spleen Effective Pyrimethamine Gaafar et al., 2014
37 Pyrimethamine/sulfadiazine Female C57BL/6 mice ME49 Chronic 20 cysts i.p Pyrimethamine, sulfadiazine 4, 100 mg/kg daily for one month Histology, qPCR, measured KP metabolites Significant increases in these kynurenine pathway metabolites were observed in the brain at 28 days post-infection Effective – Notarangelo et al., 2014
38 Pyrimethamine-loaded lipid-core nanocapsules Female CF1 mice RH Acute 103 tachyzoites 5.0–10 mg/kg/day Surviving mice, cyst brain evaluation, bioassay urea, AST and ALPo Survival rate higher than the animals treated with the same doses of non-encapsulated pyrimethamine Effective – Pissinate et al., 2014
39 Atovaquone and astragalus combination BALB/c RH Acute 2 × 104/ml trophozoites Atovaquone, astragalus 100, 0.075 mg/kg/day oral gavage Peritoneal trophozoite numbers, IL-2, IL-12, IFN-γp levels were determined by ELISA The number of trophozoites in the combination groups were found significantly lower than the number of trophozoites in the control group Effective – Sönmez et al., 2014
40 Rolipram Female Swiss albino mice KSU strain Chronic 20 tissue cysts 10 mg/kg daily for three weeks Life expectancy, serum Alt, histopathology of liver and brain Rolipram exerts a significant lowering effect on ALT levels, pathology Partially effective – Afifi et al., 2014
41 Rolipram Female Swiss albino mice Low pathogenic strain Chronic 20 tissue cysts – Tissue injury scoring, brain cyst count, specific Ig G titers, TNF- αq, IFN- γ and IL-12 assays Significant reduction of TNFα (84.6%), IFN- γ (76.7%), IL-12 (71%) Partially effective – Afifi and Al-Rabia, 2015
42 Triclosan (TS) and triclosan-loaded liposomal nanoparticles Swiss strain Albino mice RH HXGPRT (-) Acute 104 tachyzoites 150 mg/kg TS or 100 mg/kg TS liposomes Mice mortality, peritoneal, liver parasite burdens Reduction in mice mortality, parasite burden Effective – El-Zawawy et al., 2015b
43 Sulfamethoxazole-trimethoprim (ST) associated with resveratrol Male Swiss albino mice VEG strain Chronic 50 cysts containing bradyzoites ST (groups B, F), free resveratrol (groups C,G) 0.5, 100 mg kg−1 Cyst counts in the brain, and histopathology analyses Combination was able to reduce the number of cysts in the brain, inflammatory infiltrates in the liver, prevented the occurrence of hepatocytes lesions Effective Sulfamethoxazole, trimethoprim Bottari et al., 2015
44 Ciprofloxacin derivatives (compounds 2, 4,5) Female Swiss mice RH Acute 5 × 103 tachyzoites i.p 25, 50, 100, or 200 mg/kg/day a single oral dose Survival rate, determine the serum levels of urea and creatinine kinase Increased mouse survival significantly, with 13–25% of mice surviving for up to 60 days post infection Effective – Martins-Duarte et al., 2015
45 Triclosan (TS), TS liposomal Swiss albino mice ME49 Chronic 10 cysts 200, 120 mg/kg Mortality,brain parasite burden TS significant diminution in the parasite burden, great reduction in the infectivity power of T.gondii cysts Effective – El-Zawawy et al., 2015a
46 2-(Naphthalene-2-γlthiol)-1H Indole 2-(naphhalene-2-ylthio)-1H-indole BALB/c RH Acute 2 × 106 tachyzoites exposed to the concentrations of the compound i.p. 25–800 μM for 1.5 h Surviving mice, stained by PI and analyzed by fluorescence-activated cell sorting (FACS) The longevity of mice was dose dependent. Five mice out of group 400 μmol and 3 out of group 800 μmol showed immunization to the parasite Effective – Asgari et al., 2015
47 Propranolol BALB/c RH Acute 1 × 103 tachyzoites i.p 2, 3 mg/kg/day Parasite load determined In the pre-treatment group, propranolol combined with pyrimethamine was more effective Effective Pyrimethamine Montazeri et al., 2015
48 Aripiprazole BALB/c Tehran strain Chronic 50 tissue cysts, i.p 10, 20 mg/kg Cysts counted in smears prepared from brain homogenate by optical microscope No significant difference between mean logarithms of brain cyst numbers of aripiprazole groups compared with control No effective – Saraei et al., 2015
49 Pyrimethamine (PYR) and sulphadiazine (SDZ) combined with levamisole and echinacea BALB/c RH 30 days after treatment 105 tachyzoite i.p PYR; 6.25, 12.5 SDZ; 100, 200 PYR, SDZ, levamisole; 2.5, echinacea; 130, 260 mg/kg/day oral treatment 24 h later for 10 days Survival rates Survival rate PYR+SDZ, and levamisole = 33.3% to 88.9% Effective Pyrimethamine, sulfadiazine Köksal et al., 2016
50 Miltefosine Swiss albino mice RH ME49 Acute, chronic 2500 tachyzoites i.p 10 cysts orally 20 mg/kg for 5 days Survival rates, tachyzoites count in the liver, spleen, cyst count and size in the brain ultra structural study, and histopathological study Survival rate in acute = 30% Survival rate in chronic = 5% No effective in acute. Partially effective in chronic Sulphadizine Eissa et al., 2015
20 mg/kg/day
60 days post infection for 15 days
51 Tetraoxanes Female Swiss Webster RH Acute 102 and 106tachyzoite i.p 10 mg/kg/day, subcutaneously for 8 days Survival rates and pathohistological analysis Survival rate = 20 % Effective – Opsenica et al., 2015
52 Guanabenz BALB/c ME49Prugniaud Acute, chronic 104 ME49or 106 Pru tachyzoites, i.p 5 or 10 mg/kg repeated every 2 days Survival of mice, qPCR Enhanced survival, reduces cyst burdens in chronically infected mice Effective – Benmerzouga et al., 2015
53 Fluphenazine and Thioridazine BALB/c Tehran strain chronic 20 tissue cysts i.p Thioridazine 10, 20, fluphenazine 0.06 mg/kg/ three days after inoculation for 3 weeks The number of brain cysts Drugs reduced the percent of cysts at higher dose compared to lower doses Effective, not significant Pyrimethamine Saraei et al., 2016
54 Nitrofurantoin Female ICR mice RH Acute 1 × 105 tachyzoites 20, 50, and 100 mg/kg, orally once/day for 4 days The numbers of tachyzoites in the peritoneal cavity, Hematology and biochemical parameters The inhibition rate = 44.7% hematology indicators and biochemical parameters reduced by nitrofurantoin significantly Effective Pyrimethamine Yeo et al., 2016
55 Dextran sulfate Pigs RH Acute 1 × 106 tachyzoites, intravenously 50–500 μg per head host clinical, pathological, and immunological analyses High-dose caused reversible hepatocellular degeneration of the liver Effective . Kato et al., 2016
56 Propranolol BALB/c RH Acute, chronic 1 × 103 tachyzoites i.p 2, 3 mg/kg/day Parasite load determined by qPCR, and survival rate Decreased the parasite load in brain, eye, and spleen tissues Effective Pyrimethamine Montazeri et al., 2016
57 Resveratrol and sulfamethoxazole-trimetropim Male Swiss Webster VEG Chronic 50 cysts orally Oral doses of 0.5 and 100 mg/kg/day Counting brain cysts, tissue oxidant and antioxidant levels, and histopathology A reduction on the number of cysts in the brain was observed Co-administration more effective Sulfamethoxazole-trimethoprim Bottari et al., 2016
58 Compound 22 of sulfur-containing linear bisphosphonates Webster mice RH Acute 20 or 100 or 5000 tachyzoites i.p 0.05, 0.1, 0.5, and 1 mg/kg of 22/ i.p. for 10 days Survival rate ED50= 0.02 mg/kg Effective . Szajnman et al., 2016
59 Compound32 (TgCDPK1 inhibitor) Female CF-1 CBA/J RH ME49 Acute, chronic less than 100 tachyzoites/mL 20 mg/kg for The numbers of tachyzoites in spleen, brain/ and the number of brain cysts Reducing infection in spleen and brain (99%, 95%) 88.7% reduction of brain cyst Effective . Vidadala et al., 2016
5 days/ oral gavage
30 mg/kg for 14 days

2-hydroxy-3-(1_-propen-3-phenyl)-1,4-naphthoquinone.

Indirect immunofluorescence antibody test.

Reverse transcription polymerase chain reaction.

Polymerase chain reaction.

Quantitative Polymerase chain reaction.

Fluconazole.

Itraconazole.

1-Hydroxy-2-Alkyl-4(1H) Quinolone.

6-trifluoromethyl-2-thiouracil.

3-[{2-((E)-furan-2-ylmethylene) hydrazinyl} methylene]-1, 3-dihydroindol-2-one.

Lipid peroxidation.

Glutathione-S-transferase.

Alanine aminotransferase.

Aspartate amino transferase.

Alkaline phosphatase.

Interferon gamma.

Tumor necrosis factor.

 Cell culture

The cell cultures used in in vitro studies were mostly human foreskin fibroblast (HFF; 39 studies), LLCMK2 (12 studies), Vero (11 studies), Hela (6 studies), mouse macrophage cell line (J774A.1) (5 studies), and MRC-5 (2 studies; Table 3).

 Laboratory animals

T. gondii can infect most warm-blooded animals, and is studied in different animal models depending on the nature of the investigation (Szabo and Finney, 2016). The animal model used in studies was mostly mice (16 studies BALB/c and 19 studies Swiss-Webster). In murine models of acute toxoplasmosis, some medicines were protective even when administered at low dosages. But some drugs despite of their excellent in vitro activity were poorly protective in murine models with acute toxoplasmosis (Payne et al., 2013).

 Diagnostic tests and evaluation methods

The present review outlines the results of in vitro screening methods including morphological assay, incorporation of [3H] uracil assay, plaque assays, enzyme-linked immunosorbent assay (ELISA), colorimetric micro titer assay (b-galactosidase assay), flow cytometric quantification assay, and cell viability assay. Numerous versions of fluorescent proteins have been expressed in T. gondii (Kim et al., 2001). The reporter genes used in vitro and in vivo studies were the green fluorescent protein (GFP) and yellow fluorescent protein (YFP). Parasites expressing fluorescent proteins can also be analyzed and sorted by flow cytometry. This technology used for drugs screening in 10 studies.

Details about the diagnostic methods and drug dosage under in vivo conditions are shown in Table 4. Also, a comprehensive list of drugs/compounds evaluated against T. gondii with regard to IC50 is illustrated in Table 5.

A comprehensive list of drugs/compounds evaluated against T. gondii with regard to IC50.

Drug IC50 (μM) References
<1 1–5 5–10
Novel quinuclidine + Martins-Duarte et al., 2006
Novel ferrocenic atovaquone derivatives Atovaquone (PLK strain) 2d, 2e, 2f Baramee et al., 2006
SAHAa, SBHAb, Scriptaid, Trichostatin A Scriptaid Strobl et al., 2007
Trichostatin A
SAHA
SBHA
Pyridinylimidazoles SB203580 and SB202190 RWJ67657, (ME49 strain) SB202190 SB203580 Wei et al., 2007
SB203580
RWJ68198, (ME49 strain) RWJ68198, (RH strain)
RWJ67657, (RH strain)
1-hydroxy-2-dodecyl-4(1H) quinolone + Saleh et al., 2007
Fluorine-containing aryloxyethyl thiocyanate derivatives Compound 1, 3, 9 Compound 10 Liñares et al., 2007
Novel diamidine analog + Leepin et al., 2008
Pyrimethamine, sulfadiazine, atovaquone + Meneceur et al., 2008
Novel triazine JPC-2067-B + Mui et al., 2008
2-alkylaminoethyl-1,1-bisphosphonic acids Compound 19 Compound 14, 17 Szajnman et al., 2008
Itraconazole + Martins-Duarte Edos et al., 2008
Thiolactomycin analog Compound 5, 6 Compound 2 Martins-Duarte et al., 2009
Fluconazole (FLZ) FLZ (48 h) FLZ (24 h) Martins-Duarte et al., 2010
1-Hydroxy-2-Alkyl-4(1H) Quinolone derivatives + Bajohr et al., 2010
Haloperidol, clozapine, fluphenazine, trifluoperazine, thioridazine + Goodwin et al., 2011
Novel azasterols Compound 3 (48 h) Compound 1 (48 h), 2, 3 (24 h) Compound 1 (24 h) Martins-Duarte et al., 2011
Endochin-like quinolones + Doggett et al., 2012
Pterocarpanquinone + Portes Jde et al., 2012
New naphthoquinones (QUI), an alkaloid QUI-11 Ferreira et al., 2012
Liriodenine
Di-cationic, pentamidine-analog + Kropf et al., 2012
Fuconazole combined with sulfadiazine and pyrimethamine Pyrimethamine + Martins-Duarte et al., 2013
Antipsychotic drugs and valproate Fluphenazine Zuclopenthixol Fond et al., 2014
Thioridazine
Fusidic acid + Payne et al., 2013
Ivermectin and sulphadiazine Ivermectin Sulphadiazine Bilgin et al., 2013
Novel ruthenium complexes,(compounds 16 and 18) + Barna et al., 2013
Auranofin + Andrade et al., 2014
6-Trifluoromethyl-2-thiouracil + Choi et al., 2014
200 drug-like, 200 probe-like compounds of Malaria Box MMV007791 MMV007881 Boyom et al., 2014
MMV007363
MMV006704
MMV666095
MMV020548
MMV085203
Quinoline derivatives 8-Hydroxyquinoline, A 11, A14, A18, B11, B12, B15, B23, B24 A2-6, A12, A15—17, A23, B16, B22, B26, B27, B29, Chloroquine Quinoline Kadri et al., 2014
2-chloroquinoline
5-Nitroqu
Inoline Quinoline
N-oxide hydrate A7, B18
Bumped Kinase Inhibitor 1294 + Doggett et al., 2014
Salicylanilides 3i, 3j, 7a, 14a, 14b Fomovska et al., 2012
Antiretroviral compounds Atazanavir Fosamprenavir Monzote et al., 2013
Ritonavir Nelfinavir
Saquinavir
Spiroindolone + Zhou et al., 2014
Ciprofloxacin derivatives Compound 2, 5 Compound 4 Dubar et al., 2011
Thiazolidin-4-one derivatives 12A 27, 34 A 36 A D'Ascenzio et al., 2014
N6-benzyladenosine analog Compound 11 e, g, j, n, o, q, u, v Kim et al., 2007
Naphthoquinone derivative LQB151 (48 h) LQB94 da Silva et al., 2015
LQB151 (24 h)
LQB150 (24, 48 h)
Oryzalin analogs Compound 6a, h, i, 14a, 18a, b, c Compound 6b, g, j, I, n, 12 Compound 6m, 14b Endeshaw et al., 2010
94 compounds + Dittmar et al., 2016
6-(1,2,6,7-tetraoxaspiro[7.11] nonadec-4-yl)hexan-1-ol (N-251) + Xin et al., 2016

Suberoylanilide hydroxamic acid.

Suberic bishydroxamic acid.

 Discussion

The aim of this systematic review was to investigate the in vitro and in vivo effects of anti-Toxoplasma drugs and synthetic compounds, from 2006 to 2016. The current anti-T. gondii chemotherapy is deficient; as it is not well-tolerated by immunocompromised patients and cannot completely eradicate tissue cysts produced by the parasite (Rodriguez and Szajnman, 2012). Therefore, developing new, safe, effective, and well-tolerated drugs with novel mechanisms of action could be a global priority (Lai et al., 2012). An ideal drug for prophylaxis and/or treatment of toxoplasmosis would show effective penetration and concentration in the placenta, transplacental passage, parasiticidal properties vs. the different parasitic stages, penetration into cysts, and distribution in the main sites. No available drug fulfills these criteria (Derouin et al., 2000; Montoya and Liesenfeld, 2004).

Thus, the findings of the present systematic review article encourage and support more accurate investigations for future to select new anti-Toxoplasma drugs and strategies in designing new targets with specific activity against the parasite.

Activities of anti-<italic>toxoplasma</italic> clinically available drugs

With growing parasite resistance to therapeutic drugs and in the absence of a vaccine, to increase the effectiveness of drugs, various changes have been made in construction of the clinically available medicines. Thus, the activity of new formulations of clinically available drugs against T. gondii should be evaluated to find alternative treatments for toxoplasmosis (da Cunha et al., 2010).

Interestingly, encapsulation of pyrimethamine improved the efficacy and tolerability of this drug against acute toxoplasmosis in mice and can be considered as an alternative for reducing the dose and side effects of pyrimethamine (Pissinate et al., 2014). Recently, researchers reported that computational analysis of biochemical differences between human and T. gondii dihydrofolate reductase enabled the design of inhibitors with both improved potency and selectivity against T. gondii (Welsch et al., 2016). El-Zawawy et al. reported that incorporating triclosan into in the lipid bilayer of liposomes allowed its use in lower doses, which in turn, reduced its biochemical adverse effects (El-Zawawy et al., 2015b). In another study, sodium dodecyl sulfate (SDS)-coated atovaquone nanosuspensions (ANSs) considerably increased the therapeutic efficacy against experimentally reactivated and acquired toxoplasmosis by improving passage of gastrointestinal and blood-brain barriers. Accordingly, coating of ANSs with SDS may improve the treatment of toxoplasmic encephalitis and other cerebral diseases (Shubar et al., 2011).

Also, various studies showed that a number of drugs were investigated for the mechanisms of action summarized in Table 2 and Figure 2. One study discussing the metabolic differences between the host and the parasite noted that dihydrofolate reductase, isoprenoid pathway, and T. gondii histone deacetylase are promising molecular targets (Rodriguez and Szajnman, 2012).

Novel triazine JPC-2067-B (4, 6-diamino-1, 2-dihydro-2, 2-dimethyl-1-(3′(2-chloro-, 4-trifluoromethoxyphenoxy)propyloxy)-1, 3, 5-triazine), the anti-folate medicines, is highly effective against T. gondii with an IC50 of 0.02 μM, which is more efficacious than pyrimethamine and has in vitro cidal activity. Additionally, pro-drug JPC-2056 (1-(3′-(2-chloro-4-trifluoromethoxyphenyloxy) propyl oxy)-5-isopropylbiguanide) is effective in vivo when administered orally (Mui et al., 2008). Moreover, histone deacetylase is potentially a very important drug target in T. gondii, since scriptaid and trichostatin A had the highest effect against T. gondii tachyzoite proliferation with the IC50 of 0.039 and 0.041 μM, respectively (Strobl et al., 2007). For promising anti- T. gondii drugs/compounds, assessment of their ability to control parasite growth is a key step in drug development (McFarland et al., 2016).

A large number of research papers suggested that the apicoplast represents a potential drug target for new chemotherapy, as it is essential to the parasite and it is absent in host cells. Functions of the apicoplast include fatty acid synthesis, protein synthesis, DNA replication, electron transport, and heme biosynthesis (Yung and Lang-Unnasch, 2004). Some of the drugs evaluated against T. gondii are shown to act in the apicoplast such as thiolactomycin, triclosan (TS), azithromycin, fusidic acid, ciprofloxacin, and quinoline derivatives (Costa et al., 2009; Martins-Duarte et al., 2009, 2015; Payne et al., 2013; Kadri et al., 2014; El-Zawawy et al., 2015b).

In T. gondii, FAS-II enzymes are present in the apicoplast and are essential for its survival. The key enzyme in this process is the ENR enzyme, which is not found in mammals (Surolia and Surolia, 2001). This enzyme catalyzes the last reductive step of the type II FAS pathway. The TS, which inhibits type II FAS, significantly reduced mice mortality, parasite burden, as well as viability and infectivity of tachyzoites and cysts harvested from infected treated mice and their brains. Accordingly, TS is proved as an effective, promising, and safe preventive drug against acute and chronic murine toxoplasmosis. Liposomal formulation of TS enhanced its efficacy and allowed its use at a lower dose (Surolia and Surolia, 2001; El-Zawawy et al., 2015a,b). Among apicoplast pathways, DNA replication is an important potential chemotherapeutic target. Fluoroquinolones are the known DNA replication inhibitors that target prokaryotic type II topoisomerases (Collin et al., 2011). In two studies, researchers showed that derivatives of the antibiotic ciprofloxacin, a fluoroquinolone, are active against T. gondii tachyzoites both in vitro and in vivo (Neville et al., 2015). While all mice treated with ciprofloxacin died by day 10 post-infection, some mice treated with ciprofloxacin derivatives remained alive for at least 60 days, suggesting that ciprofloxacin derivatives cured T. gondii infection in treated mice (Dubar et al., 2011; Martins-Duarte et al., 2015).

 Anti-<italic>toxoplasma</italic> activities of new synthetic compounds

There are numerous reports on efficacy of many new synthetic compounds with a focus on identifying drug candidates with innovative and acceptable profiles against T. gondii. The anti-coccidial effect of 1-[4-(4-nitrophenoxy) phenyl] propane-1-one (NPPP), a synthetic compound, was studied both in vitro and in vivo. Treatment with NPPP showed anti-Toxoplasma activity in vitro with a lower EC50 value than pyrimethamine. In ICR mice infected with T. gondii, oral administration of NPPP for 4 days showed statistically significant anti-Toxoplasma activity with lower number of tachyzoites than those of the negative control (Choi et al., 2015).

In a study by Kadri et al. anti-Toxoplasma properties of 58 newly synthesized quinoline compounds were evaluated. A significant improvement in anti-Toxoplasma effect among quinoline derivatives was detected in B11, B12, B23, and B24. Among these compounds, B23 was the most effective compound with the IC50 value of < 1 μM, displaying its anti-Toxoplasma effects and ability to cause the disappearance of the apicoplast (40–45% of the parasites lost their apicoplasts; Kadri et al., 2014).

In a study by Boyom et al. the strategy adopted was to repurpose the open access Malaria Box to identify chemical series active against T. gondii. The results showed that the most interesting compound was MMV007791, a piperazine acetamide, which has an IC50 of 0.19 μM. This compound is novel for its anti-Toxoplasma activity, and of course, further studies on the rates and mechanisms of compound action will elucidate these considerations (Boyom et al., 2014).

Tetraoxanes, anti-cancer molecules, were tested in vivo against T. gondii. Subcutaneous, administration of a 10 mg/kg/day dose of derivative 21, for 8 days allowed the survival of 20% of infected mice, demonstrating the high potential of tetraoxanes for the treatment of T. gondii (Opsenica et al., 2015).

In another study by Moine et al. researchers evaluated in vitro anti-T. gondii activity of 51 compounds with a biphenylimidazoazine scaffold. Eight of these compounds displayed highly potent activity against T. gondii growth in vitro, with 50% effective concentration (EC50) below 1 mM, without demonstrating cytotoxic effects on human fibroblastic cell at equivalent concentrations. However, these compounds have to be evaluated in animal models so as to confirm their in vivo activity (Moine et al., 2015a).

Several pathways were characterized and shown to differ significantly from those of the mammalian host cells, thus, revealing an attractive area for therapeutic intervention. 1-Hydroxy-2-Alkyl-4 (1H) quinolone derivatives inhibit the fourth step of the essential de novo synthesis of pyrimidine, which uses ubiquinol reduction as an electron sink for dihydroorotate oxidation (Saleh et al., 2007). Also, newly synthesized bisphosphonates interfere with the mevanolate pathway, which leads to the synthesis of sterols and polyisoprenoid compounds that are important for parasite survival (Shubar et al., 2008).

Interestingly, Kamau et al. identified novel kinases that are integral to essential pathways, elucidating their mechanism of action and ultimately, identifying new drug targets (Kamau et al., 2012). In that study, 527 compounds were evaluated in vitro; also, they assessed the impact of the inhibitory compounds C1, C2, C3, and C5 in mouse models of toxoplasmosis. C2 was found quite effective in decreasing the parasite burden and increasing mice survival. These results should be considered with caution, since there are a number of factors are at play in whether a compound will be in vivo effective, such as solubility in vivo, access to different tissues, and host metabolic processes (Kamau et al., 2012). In a recent study, Dittmar et al. screened a collection of 1,120 compounds, 94 of which were blocked parasite replications with IC50 of <5 μM. These data suggest that tamoxifen restricts Toxoplasma growth by inducing xenophagy or autophagic destruction of this parasite (Dittmar et al., 2016). According to a new study, in silico screening is useful, particularly in the identification of molecular targets in the laboratory. Fernandez et al. synthesized VAM2 compounds (7-nitroquinoxalin-2-ones), based on the design obtained from an in silico prediction with the software TOMOCOMD-CARDD. From the group of VAM2 compounds, Fernandez et al. chose VAM2-2 with an IC50 of 3.3 μM against T. gondii. However, more studies are required to evaluate its effect on the cysts formed by of the parasite and in animal models of toxoplasmosis (Fernández et al., 2016).

 Activity of drugs, compounds, and combined therapy against cysts

An ideal drug against toxoplasmosis should not only be effective against the proliferative stage of the parasite but also exert dual activity against the tissue cyst stage and penetration into cysts (Benmerzouga et al., 2015). Currently, there is no approved therapy that eliminates the tissue cysts responsible for chronic infection (Innes, 2010). Derouin reported that among the drugs commonly used in humans, only atovaquone and azithromycin were found effective after long-term incubation. Besides, arpinocid-N-oxyde, an anticoccidial for veterinary use, was efficient at a high dosage (Derouin, 2005).

Recently, investigators have focused on guanabenz for in vivo studies, as guanabenz inhibitor of eIF2a dephosphorylation, is already an food and drug administration (FDA) approved drug and has excellent solubility with good penetration into the CNS. The results of that study show that guanabenz (5 mg/kg/day) not only protects mice against acute toxoplasmosis, but also reduces 69% of the number of brain cysts in chronically infected animals. This finding suggested that guanabenz can be repurposed into an effective antiparasitic with a unique ability to diminish tissue cysts in the brain (Benmerzouga et al., 2015).

Another study showed that miltefosine had no efficacy in controlling acute toxoplasmosis after 5 days of treatment; however, a 15-day treatment against the established chronic stage led to a 78% reduction of cysts in the brain. Additionally, the remaining cysts were noticeably smaller upon microscopic examination, suggesting that the drug effectively penetrates the blood-brain barrier, and that extension of treatment time may produce greater effects (Eissa et al., 2015).

In another study by Maubon et al. FR235222 and its derivatives were identified as new lead compounds for use against acute and chronic toxoplasmosis both in vitro and in vivo. In vivo experiments indicated that FR235222, as a histone deacetylase inhibitor, is able to access the bradyzoites within the cyst. The ability of FR235222 to permeate the membrane wall is a major advantage for crossing the blood-brain barrier and CNS tissue, where Toxoplasma cysts are located. This opens a promising way to develop drugs that are selective against Toxoplasma and those that have sterilizing activity, especially in patients with cysts, who are at risk for reactivating acute toxoplasmosis (patients with HIV infection, hematological malignancies, or transplantation). Still, effectiveness of FR235222 against chronically infected mice remains to be directly demonstrated in vivo (Maubon et al., 2010).

In a new study Vidadala et al. identified compounds 32 (T. gondii calcium-dependent protein kinase 1 inhibitor) a promising lead for the development of a new antitoxoplasmosis therapy. Compounds 32 is CNS-penetrant and highly effective in acute and latent mouse models of T. gondii infection, significantly reducing brain cysts by 88.7% (Vidadala et al., 2016).

Many studies reported anti- Toxoplasma effects of different drugs combination with novel compounds. The compound 2-hydroxy-3-(1′-propen-3-phenyl)-1, 4-naphthoquinone (PHNQ6), (50 mg/kg/day) combined with sulfadiazine showed reduction or elimination of brain cysts in vivo (Ferreira et al., 2006). In another study that coadministered spiramycin and metronidazole, spiramycin, did not reach effective concentrations in the brain due to the presence of the efflux transporters multidrug-resistant protein 2, and P-glycoprotein. Metronidazole increased brain penetration of spiramycin, causing a significant reduction of T. gondii brain cysts, with potential clinical translatability for chronic toxoplasmosis treatment. According to the reports, combination therapy leads to faster recovery, less relapse, lower doses of drugs, and fewer side effects of the disease. Furthermore, such combinations are highly promising to develop a drug that is able to eliminate the cyst stage of the parasite, and thus, efficiently impairs relapse of the disease (Chew et al., 2012; Martins-Duarte et al., 2013).

 Activity of drugs against congenital toxoplasmosis

In pregnant women, current toxoplasmosis treatment is based on the administration of spiramycin or a drug combination such as sulphadiazine-pyrimethamine-folinic acid (SPFA) in cases of confirmed fetal infection. However, these drugs are not well-tolerated and present many adverse effects due to their toxic effects to the host (Degerli et al., 2003).

Degerli et al. evaluated the effectiveness of azithromycin, artemisia annua infusion, spiramycin, and SPFA in Calomys callosus, such as model of congenital toxoplasmosis. The results demonstrated that the treatment of pregnant C. callosus with azithromycin was effective for inhibiting the vertical transmission of T. gondii ME49 strain, suggesting that it may be an alternative drug of choice for the treatment of congenital infection, since it is able to inhibit fetal infection and offers new perspectives for the treatment of congenital toxoplasmosis. Azithromycin is one of the new generation macrolides with numerous advantages. Mechanism of action of azithromycin is based on the inhibition of protein synthesis in both T. gondii tachyzoite and bradyzoite stages (Degerli et al., 2003), but it may present limited effectiveness against T. gondii, requiring high drug concentrations (Costa et al., 2009). In another study, Oz et al. reported that combined atovaquone and diclazuril therapy is a novel synergistic prophylactic and therapeutic approach to fetal maternal toxoplasmosis (Oz, 2014a). Atovaquone, an inhibitor of mitochondrial electron-transport processes, is an FDA-approved toxoplasmosis therapy but not for use in congenital toxoplasmosis treatment (Oz, 2014a). Another compound, diclazuril, and its related benzeneacetonitriles have long been used in the treatment and prevention of poultry and livestock coccidiosis. In addition, it is known to be a safe compound at therapeutic dose levels (Assis et al., 2010).

 Adverse effects of drugs

However, anti-Toxoplasma effects of drugs/compounds were reported in many trials, but prednisolone increased the number of tachyzoites and bradyzoites in immunosuppressed infected mice (Puvanesuaran et al., 2012). In addition, betamethasone can escalate the invasion of tachyzoites, in cell culture. It could be suggested that patients under prolonged use of betamethasone and prednisolone should be protected against T. gondii infection. Also, if individuals receiving betamethasone are infected with T. gondii, interferon-gamma may be used to reduce the invasion of tachyzoites (Ghaffarifar et al., 2006).

 Conclusions

As current chemotherapy against toxoplasmosis is still not satisfactory, the development of well-tolerated and safe specific immunoprophylaxis in relaxing the need of dependence on chemotherapeutics is a highly valuable goal for global disease control. Immunotherapeutics strategies for improving toxoplasmosis control could either be a vaccine which would induce strong protective immunity against toxoplasmosis, or passive immunization in cases of disease recrudescence. However, with the increasing number of high-risk individuals, such as immunocompromised patients, and absence of a proper vaccine, continued efforts are necessary for the development of novel treatment options against T. gondii. Some of the novel compounds reviewed here may represent good starting points for the discovery of effective new drugs. In further bioinformatic and in silico studies are needed in order to identify new potential toxoplasmicidal drugs.

 Author contributions

AD and MS conceived the idea for this review. MM and SS searched the databases for potentially eligible articles based on their titles and abstracts. AD and MM participated in the study design and wrote the manuscript. SM and EA critically reviewed the manuscript. All authors read and approved the final manuscript for publication.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We also would like to thank of financial support by Vice Chancellors for Research of Mazandaran University of Medical Sciences, Sari, Iran (Grant number: 2085).

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