Synergistic Activity between Statins and Bisphosphonates against Acute Experimental Toxoplasmosis

Abstract

Bisphosphonates are widely used for the treatment of bone disorders. These drugs also inhibit the growth of a variety of protozoan parasites, such as Toxoplasma gondii, the etiologic agent of toxoplasmosis. The target of the most potent bisphosphonates is the isoprenoid biosynthesis pathway enzyme farnesyl diphosphate synthase (FPPS). Based on our previous work on the inhibitory effect of sulfur-containing linear bisphosphonates against T. gondii, we investigated the potential synergistic interaction between one of these derivatives, 1-[(n-heptylthio)ethyl]-1,1-bisphosphonate (C7S), and statins, which are potent inhibitors of the host 3-hydroxy-3-methyl glutaryl-coenzyme A reductase (3-HMG-CoA reductase). C7S showed high activity against the T. gondii bifunctional farnesyl diphosphate (FPP)/geranylgeranyl diphosphate (GGPP) synthase (TgFPPS), which catalyzes the formation of FPP and GGPP (50% inhibitory concentration [IC₅₀] = 31 ± 0.01 nM [mean ± standard deviation]), and modest effect against the human FPPS (IC₅₀ = 1.3 ± 0.5 μM). We tested combinations of C7S with statins against the in vitro replication of T. gondii We also treated mice infected with a lethal dose of T. gondii with similar combinations. We found strong synergistic activities when using low doses of C7S, which were stronger in vivo than when tested in vitro We also investigated the synergism of several commercially available bisphosphonates with statins both in vitro and in vivo Our results provide evidence that it is possible to develop drug combinations that act synergistically by inhibiting host and parasite enzymes in vitro and in vivo.

Keywords: Toxoplasma gondii; bisphosphonate; isoprenoids; statins; synergy.

FIG 1

Structures of the compounds discussed in this work.

FIG 2

Effects of various doses of C7S (A) or atovaquone (B) on survival of mice treated with a lethal dose of RH tachyzoites (infection). Treatment was once a day for 10 days starting 6 h after infection. The ED₅₀s calculated were 0.87 mg/kg/day for C7S and 2.53 mg/kg/day for atovaquone. The graphs show the combination of the results of four individual experiments, shown in Fig. S2 (C7S) and Fig. S3 (atovaquone) in the supplemental material. For each experiment, the controls received only the drug solvent (10% Kolliphor HS 15) instead of the treatment. As expected, all control animals succumbed to the infection at 9 to 11 days. Statistical analysis using the Kaplan-Meier log rank test (GraphPad Prism 7 software) indicated significant differences (P < 0.0001) between the results for the control animals (untreated) and those that received various doses of C7S or atovaquone. The number next to each line indicates the dose in mg/kg/day.

FIG 3

Effects of various combinations of C7S and atorvastatin on survival of mice infected with tachyzoites. (A) Mouse survival after treatment with atorvastatin (ATV) (16.2 mg/kg/day) and C7S (0.44 mg/kg/day) as single drugs or in combination (ATV + C7S) for 10 days using 5 mice/experimental group. Results are from 2 or 3 experiments. (B) Survival curves after treatment with various doses of C7S and atorvastatin (0.075, 0.15, 0.25, and 0.5× ED₅₀); the ED₅₀s were 0.87 mg/kg/day and 32.4 mg/kg/day, respectively. Results are from 2 to 4 experiments using 5 mice per combination. Mice in the control groups received only 10% Kolliphor HS 15 and, as expected, succumbed to the infection at 9 to 10 days. (C) Data from panel B and 0.02× ED₅₀s were used to calculate the combination index (CI) values, and the results graphed against the total drug doses (atorvastatin + C7S). Statistical analysis using the Kaplan-Meier log rank test indicated significant differences (P < 0.0001) between the results for the control (untreated) animals and those that received C7S or atorvastatin together and each drug separately or controls (A) and the different drug combinations compared to controls (B).

FIG 4

Effects of combinations of atorvastatin (ATV) with several bisphosphonates on survival of mice infected with tachyzoites. (A) Groups of mice were either untreated or treated with zoledronate alone (0.01 mg/kg/day) or the combination of zoledronate (0.01 mg/kg/day) and atorvastatin (1.25 mg/kg/day). (B) Survival at 30 days of mice treated with atorvastatin (1.25 mg/kg/day) or zoledronate (0.01 mg/kg/day) alone or both in combination. (C) Survival at 30 days of mice treated with atorvastatin (1.25 mg/kg/day) or risedronate (0.01 mg/kg/day) alone or both in combination. (D) Survival at 30 days of mice treated with atorvastatin (1.25 mg/kg/day) or C7S (0.03 mg/kg/day) alone or both in combination. In all cases, treatment was started 6 h after initial infection and administered daily for 10 days, and 5 mice per group were used. The results for each combination are from two independent experiments. Statistical analysis using the Kaplan-Meier log rank test indicated significant differences (P < 0.0001) between the results for control animals (untreated) and animals treated with the different drug combinations (A to D). Error bars show standard deviations.