Efficacy of Guanabenz Combination Therapy Against Chronic Toxoplasmosis Across Multiple Mouse Strains

Abstract

21Toxoplasma gondii, an obligate intracellular parasite that can cause life-threatening 22 acute disease, differentiates into a quiescent cyst stage to establish lifelong chronic infections in 23 animal hosts, including humans. This tissue cyst reservoir, which can reactivate into an acute 24 infection, is currently refractory to clinically available therapeutics. Recently, we and others have 25 discovered drugs capable of significantly reducing brain cyst burden in latently infected mice, 26 but not to undetectable levels. In this study, we examined the use of novel combination 27 therapies possessing multiple mechanisms of action in mouse models of latent toxoplasmosis. 28Our drug regimens included combinations of pyrimethamine, clindamycin, guanabenz, and 29 endochin-like quinolones (ELQs), and were administered to two different mouse strains in an 30 attempt to eradicate brain tissue cysts. We observed mouse strain-dependent effects with these 31 drug treatments: pyrimethamine + guanabenz showed synergistic efficacy in C57BL/6 mice, yet 32 did not improve upon guanabenz monotherapy in BALB/c mice. Contrary to promising in vitro 33 results demonstrating toxicity to bradyzoites, we observed an antagonistic effect between 34 guanabenz + ELQ-334 in vivo. While we were unable to completely eliminate brain cyst burden, 35we found that a combination treatment of ELQ-334 + pyrimethamine impressively reduced brain 36 cysts to 95% in C57BL/6 mice, which approaches the limit of detection. These analyses 37 highlight the importance of evaluating anti-infective drugs in multiple mouse strains and will help 38 inform further preclinical cocktail therapy studies designed to treat chronic toxoplasmosis. 39 40

Factors influencing tissue cyst yield in a murine model of chronic toxoplasmosis

“…Drug efficacy is currently evaluated exclusively based on tissue cyst recovery which has a sensitivity limited to under two orders of magnitude. Thus, even the most effective experimental drugs which reduce recovery by between 80-90% (10-13, 15, 16), fall within the range of cyst recovery in the absence of any drug treatment (5, 9) (and data presented in this study). We therefore need to reassess overall approaches to understanding the chronic infection and focus at the level of encysted bradyzoites, appreciating the heterogeneity of activity within the tissue cyst.…”

“…A fundamental limitation in the evaluation of potential drugs effective in the chronic infection is the fact that efficacy measured by the apparent reduction in cyst burdens following treatment are presented in percentage terms (10, 12, 13, 15, 16, 58). While this level of sensitivity represents the currently acceptable norm, they fail to address the mechanistic basis for drug susceptibility or resistance.…”

“…A fundamental limitation in the evaluation of potential drugs effective in the chronic infection is the fact that efficacy measured by the apparent reduction in cyst burdens following treatment are presented in percentage terms (10,12,13,15,16,58).…”

Endochin-like quinolones (ELQs) and bumped kinase inhibitors (BKIs): Synergistic and additive effects of combined treatments against Neospora caninum infection in vitro and in vivo

“…Thus, it will be important in further studies to investigate the adjustment of the dosages, taking into account the results presented here: while we had shown previously that ELQ-334 administered at 10 mg/kg/day led to a 50% reduced vertical transmission ( Anghel et al, 2018 ), we here show that a lower dose of 5 mg/kg/day inhibits pup infection by > 90%, and in combination with BKI-1748 at 20 mg/kg, vertical transmission is completely blocked. It is noteworthy to mention that a combination of ELQ-334 and another cytochrome bc1 inhibitor, atovaquone, resulted in complete cure of experimental Babesia microti infection in immunodeficient mice ( Lawres et al, 2016 ), and treatments of persistently T. gondii infected mice with ELQ-334 resulted in a dramatic reduction of tissue cyst in the brain and the combination of ELQ-334 with pyrimethamine further reduces the number of brain cysts ( Doggett et al, 2020 ; Martynowicz et al, 2020 ).…”

A potent HDAC inhibitor blocks Toxoplasma gondii tachyzoite growth and profoundly disrupts parasite gene expression

“…Also, the compound may not be active against the bradyzoite stage that divides at a slower pace than tachyzoites. There are currently no drugs available to treat established bradyzoite infection and only a few compounds have been shown to reduce the cyst burden [35,36]. There is a need to develop new in vitro systems that would enable the testing of compounds against bradyzoites.…”