Effect of α-Tocopheryloxy Acetic Acid on the Infection of Mice with Plasmodium berghei ANKA In Vivo and Humans with P. falciparum In Vitro

Malarial parasites are susceptible to oxidative stress. The effects of α-tocopheryloxy acetic acid (α-TEA), a vitamin E analog, on infection by Plasmodium berghei ANKA and P. falciparum in mice and human red blood cells (RBCs), respectively, were examined in this study. For in vivo studies in mice, RBCs infected with P. berghei ANKA were inoculated via intraperitoneal injection and α-TEA was administered to C57BL/6 J male mice after infection. The blood–brain barrier (BBB) permeability was examined by Evans blue staining in experimental cerebral malaria at 7 days after infection. The in vitro inhibitory effect of α-TEA on P. falciparum 3D7 (chloroquine-sensitive strain) and K1 (multidrug-resistant strain) was tested using a SYBR Green I-based assay. When 1.5% α-TEA was administered for 14 days after infection, 88% of P. berghei ANKA-infected mice survived during the experimental period. Nevertheless, all the control mice died within 12 days of infection. Furthermore, the Evans blue intensity in α-TEA-treated mice brains was less than that in untreated mice, indicating that α-TEA might inhibit the destruction of the BBB and progression of cerebral malaria. The in vitro experiment revealed that α-TEA inhibited the proliferation of both the 3D7 and K1 strains. This study showed that α-TEA is effective against murine and human malaria in vivo and in vitro, respectively. Although α-TEA alone has a sufficient antimalarial effect, future research could focus on the structure–activity relationship to achieve better pharmacokinetics and decrease the cytotoxicity and/or the combined effect of α-TEA with existing drugs. In addition, the prophylactic antimalarial activity of premedication with α-TEA may also be an interesting perspective in the future.

In this study, to determine whether α-TEA is an appropriate antimalarial drug candidate, C57BL/6 J male mice were infected with P. berghei ANKA, a lethal strain of murine malaria and experimental cerebral malaria, and treated with α-TEA. In addition, to analyze whether the antiprotozoal effects were the result of modification of the physiology of the host or a direct effect on the protozoan, the effect of α-TEA on the growth of P. falciparum in vitro and the cytotoxic effect of α-TEA on fibroblast and red blood cells were also examined.

Materials and Methods
Experimental infection with P. berghei ANKA was carried out using C57BL/6 J male mice (Japan CLEA, Tokyo, Japan) at biosafety level 2 in a specific pathogen-free facility. The room temperature (24 ± 1 °C) and humidity (50 ± 10%) were adjusted, and the lighting was controlled (lights on from 7 AM to 7 PM). The mice were allowed free access to water and a diet (CA-1; CLEA Japan, Tokyo, Japan). The animals used in this study were handled and cared for in accordance with the "Guiding Principles for the Care and Use of Research Animals" established by Obihiro University of Agriculture and Veterinary Medicine, Japan. All animal experimental procedures were approved by the Obihiro Uni- Alpha-TEA (molecular weight 488.75 g/mol) distributed by Eisai (Eisai Co., Ltd., Tokyo, Japan) was mixed with the diet [0.75% and 1.5% (w/w) of α-TEA] and fed to 8 week-old C57BL/6 J male mice (23-25 g body weight) for 14 days after infection. Drug concentrations were determined based on the results of preliminary experiments. The day of infection was defined as Day 0. The mixed diet was outsourced to Oriental Yeast Co. Ltd. (Tokyo, Japan).
For parasite infection, 4 × 10 4 P. berghei ANKA-infected red blood cells (iRBCs) were intraperitoneally injected into mice and their survival rates and parasitemia were monitored. On day 4 post-infection, 2 μL of blood was collected from the tip of the tail, smeared on a glass slide, and stained with Giemsa (Sigma-Aldrich, Tokyo, Japan). The percentage of infected erythrocytes to total erythrocytes (parasitemia) was determined using a phase contrast microscope (DIAPHOTO-TMD300, Nikon, Tokyo, Japan). More than 1,000 red blood cells (RBCs) were counted in each mouse to assess parasitemia. On day 7 after infection, the permeability of the blood-brain barrier (BBB) was evaluated by Evans blue staining as follows: 0.1 mL of 2% (w/v) Evans blue (056-04061; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) dissolved in PBS was injected into infected mice with or without treatment with α-TEA through the tail vein. Two hours after injection the mice were sacrificed, and brain samples were collected.
Survival rates were analyzed using log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests implemented in GraphPad Prism 5. Parasitemia was analyzed using a one-way analysis of variance and Tukey's test. A value of P < 0.05 was considered statistically significant.
In vitro growth inhibition of P. falciparum 3D7 and K1 was performed using the SYBR Green I-based fluorescence assay (SYBR ® Green I Nucleic Acid Stain 10,000 × , ME, USA) as previously described [27,28]. Briefly, test compounds were prepared in complete media at eight final concentrations (two-fold serial dilution) ranging from 100 to 0.78 μM. Chloroquine diphosphate (molecular weight 515.86 g/mol; Sigma-Aldrich, MO, USA) was used as the reference drug. Before use, the parasites' life cycle was synchronized by treatment with 5% D-sorbitol to obtain ≥ 90% ring-stage parasites. Synchronous parasites (50 μL) at 0.5% parasitemia and 2% hematocrit were seeded in a 96-well plate containing 50 μL of the test compounds. The 96-well plates were incubated for 72 h at 37 °C in 5% CO 2 , 5% O 2 , and 90% N 2 . Next, 100 μL of lysis buffer containing 1 × SYBR Green I was added to each well, mixed by pipetting, and then incubated in the dark at room temperature for 2 h. Fluorescence intensities were measured using a Fluoroskan Ascent instrument (Thermo Scientific, MA, USA) at excitation and emission wavelengths of 485 and 518 nm, respectively. Cells treated with 1% (v/v) DMSO were used as a negative control, and wells containing only test compounds and erythrocytes were used to correct the background signals. The inhibition assays were performed in quadruplicate for each concentration and repeated three independent times. The half-maximal inhibitory concentration (IC 50 ) values were analyzed using nonlinear regression analysis implemented in GraphPad Prism 8.
Human foreskin fibroblast cells (HFFs) were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, MO, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (× 100, Wako, Osaka, Japan) at 37 °C and 5% CO 2 in air. A cell viability assay was used to evaluate the cytotoxic effects of the tested compounds, as previously described [27]. Briefly, the cell suspension was seeded in a 96-well plate at a density of 1 × 10 4 cells/well in DMEM with 10% FBS and incubated for 48 h at 37 °C and 5% CO 2 in air. Then, two-fold dilutions (total eight concentrations) of the test compounds in DMEM were added in quadruplicate to each well and incubated for an additional 72 h. Cell Counting Kit-8 (CCK-8) was added and incubated for an additional 3 h at 37 °C and 5% CO 2 in air, and the absorbance was measured at 450 nm. The tests were independently repeated three times. The half-maximal cytotoxic concentration (CC 50 ) values were analyzed using nonlinear regression analysis implemented in GraphPad Prism 8.
The human RBC hemolysis assay was performed as previously described [29]. Briefly, each test compound was prepared in 1 × phosphate-buffered saline (PBS) at a desired concentration in a 96-well plate, and 3% RBCs suspension in PBS was added. The plate was incubated for 3 h at 37 °C in 5% CO 2 , 5% O 2 , and 90% N 2 and then centrifuged at 1300 × g for 5 min. Finally, 100 μL of the supernatant of each mixture was transferred to a new 96-well plate, and the absorbance was recorded at 540 nm. PBS (with 1% DMSO) and RBC lysis buffer (0.83% NH 4 Cl; 0.01 M Tris-HCl, pH 7.2) were used as negative and positive controls, respectively. The hemolysis rate of RBCs was calculated using the following formula: Hemolysisrate =[(A sample -A negative control ) / (A positive control -A negative control ) × 100], where A stands for absorbance. The experiments were performed in technical quadruplicates and independently repeated three times.

Results
As shown in Fig. 1A, the effect of the administration of α-TEA on the survival of P. berghei ANKA-infected mice was remarkable. Although the parasitemia of 0.75% α-TEAtreated mice was found to be significantly lower than that of the untreated group on day 10 after infection (P < 0.05; Fig. 1B), both control and 0.75% α-TEA -treated mice died within 12 days after infection (P < 0.05; Fig. 1A). Remarkably, when 1.5% α-TEA was mixed with the diet for the 14 days following infection, 88% of the P. berghei ANKAinfected mice survived the experimental period. In fact, the proportion of parasitemia in this group of mice was significantly lower than that of the control group on days 4, 8, and 10 following infection (P < 0.05). Seven days after infection, Evans blue staining was observed to be maximal in the mice from the untreated group, while staining intensity was dramatically attenuated by 0.75 and 1.5% α-TEA treatment (Fig. 2). The in vitro effects of α-TEA are summarized in Tables 1 and 2 and are shown in Fig. 3. The proliferation of P. falciparum was inhibited by α-TEA in both 3D7 (IC 50 13.89 ± 1.16 μM) and K1 (IC 50 15.51 ± 0.56 μM) strains. The cytotoxicity of α-TEA against human foreskin fibroblast (HFF) cells showed the cytotoxic concentration CC 50 value of 80.41 ± 17.24 μM, resulting in the selectivity indexes (SI) of 5.79 and 5.18 for P. falciparum 3D7 and K1, respectively. Furthermore, at a concentration of 100 μM, α-TEA exhibited an RBC hemolysis rate of 14.70 ± 1.12%.

Discussion
This study reports a significant effect for α-tocopheryloxy acetic acid (α-TEA) on the treatment of infection by P. berghei ANKA in mice. To date, α-TEA has been studied mainly for its antitumor effect [22][23][24][25]30], but the results of this study showed an antiprotozoal effect for this compound on P. yoelii 17XL infection in mice [19]. When α-TEA was mixed with the diet and orally administered to P. yoelii 17XL-infected mice, their survival was almost completely maintained and the parasites were eradicated from their blood cells [19]. In P. berghei ANKA-infected mice, a lower dosage of α-TEA (0.75%) showed similar kinetics to control mice in both survival rate and parasitemia (Fig. 1). However, when the infected mice were treated with a higher dose of α-TEA (1.5%) for 14 days after infection, their survival was significantly higher and parasitemia was significantly lower than those in the control and 0.75% α-TEA-treated groups (Fig. 1). These data suggest that it may be possible to maintain low levels of parasitemia by increasing the α-TEA concentration in the blood stream immediately before the elevation of parasitemia after the moment of infection. It is not easy to explain what causes such a high survival rate accompanied by high parasitemia, but it is a common phenomenon in treated mice that have overcome lethality in the early or middle stages of murine malaria infection [31][32][33]. It has been reported that treatment with 50 mM α-TOS after P. berghei ANKA infection significantly increased Control 0.75% 1.5% Fig. 2 Permeability of blood-brain barrier (BBB) in mice infected with P. berghei ANKA assessed by Evans blue staining. Evans blue solution was injected to infected mice through tail vein on day 7 following infection. After 2 h following injection, mice were sacrificed and dissected. Brains were taken from untreated mice infected with P. berghei ANKA (control; upper panel) and from 0.75 and 1.5% (w/w) α-TEA-treated mice infected with P. berghei ANKA (middle and lower panels, respectively). Each brain is representative of the mean coloration of its treatment group host survival in mice [17]. The median survival time of the solvent-and 50 mM α-TOS-treated mice infected with P. berghei ANKA was 8.0 and 11.5 days after infection, respectively [17]. The effect of α-TEA on P. berghei ANKA infection was greater than that of α-TOS. In our study, approximately 90% of the infected mice treated with of 1.5% α-TEA survived throughout the experimental period (Fig. 1A). As α-TEA is not hydrolyzed and has good stability [20,21], it may be a candidate antimalarial drug. To investigate if the antiprotozoal effect of α-TEA was the result of modifying the host physiology or a direct impact on the protozoan, the effect of α-TEA on the in vitro growth of P. falciparum was examined. The resulting IC 50 values against 3D7 (13.89 ± 1.16 μM) and K1 (15.51 ± 0.56 μM) strains' infection were close to those previously reported for plasma α-TEA concentration (10 μg/ml, which is equal to 20.46 μM) on the mice orally administered with 10 mg/ day for 3 days [19], indicating that α-TEA was affecting the parasite proliferation in mice. Furthermore, the rupture of the erythrocytes, which releases hemoglobin, will cause harm to numerous vital organs, including the liver, kidney, and heart, and therefore, monitoring the hemolytic activity of the tested compound is a crucial factor in avoiding the increasing toxic effect when administered for treatment [34]. To date, no reported clear criteria about the safety threshold for hemolysis rate in any mammalian erythrocytes, including humans. Referring to the plasma concentration of α-TEA, as mentioned earlier, in vitro hemolysis test exhibited a relatively low hemolysis rate (2.15 ± 0.37% at 25 μM of α-TEA). The HFF cytotoxicity data (CC 50 80.41 ± 17.24 μM, Table 2) were also considered at a safe range based on the plasma concentration and resulting SI values. For further preclinical trails, it is essential to ensure that the dose required to kill malaria parasite is not harmful in host. In previous study for safety assessment of α-TEA in mice, there was no mortality, and no clinical signs of toxicity in the α-TEA doses of 100, 300, or 1500 mg/kg/day by daily oral gavage for 28 days. Histopathological evaluation of major organs (heart, lung, kidney, liver, spleen, jejunum, ileum, and cecum) revealed no significant α-TEA treatment-related lesions. Blood counts revealed low-grade anemia, but no other significant differences between treatment and control groups [35].
Collectively, our results indicate that the antimalarial effect of α-TEA was caused by its direct impact on parasites (Table 1 and Fig. 3). Parasites may consume α-TEA from plasma and erythrocyte membranes. Similarly, in preliminary experiments, the in vitro proliferation of trypanosomes after α-TEA treatment was significantly inhibited compared with that after control treatment (no α-TEA), suggesting that α-TEA has a direct effect on trypanosomes (Supplementary Figure). In addition, α-TEA induces the production of ROS, such as hydroxyl radicals and peroxynitrite, in protozoans [36]. Oxidative stress may inhibit parasite growth. We hypothesize that parasites exposed to α-TEA accumulate ROS by interference of this molecule with the mitochondrial redox chain and by stimulation of the intrinsic apoptotic pathway. Endothelial cells lacking mitochondrial DNA are resistant to α-TEA, both in the accumulation of ROS, induction of apoptosis, and maintenance of their angiogenic potential [37].

Conclusion
This study showed that α-TEA is effective against murine and human malaria in vivo and in vitro, respectively. Although α-TEA alone has a sufficient antimalarial effect, future research could focus on the structure-activity relationship to achieve better pharmacokinetics and decrease the cytotoxicity and/or the combined effect of α-TEA with existing drugs. In addition, the prophylactic antimalarial activity of premedication with α-TEA may also be an interesting perspective in the future.