In vitro and ex vivo antiplasmodial activity of 1-(3-benzyloxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-propan-1-one) against circulating strains of Plasmodium spp. in the state of Rondônia, Brazil

Abstract

Malaria is a disease caused by Plasmodium spp. protozoa. The ability of Plasmodium to develop resistance to current antimalarial drugs makes the study of chemotherapeutic alternatives extremely important. This study aimed to evaluate the antimalarial activity of compound 3286938 (1-(3-benzyloxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-propan-1-one), which presents in its structure a 3,4,5-trimethoxyphenyl group, in vitro, using the W2 strain of P. falciparum and against circulating strains of P. vivax and P. falciparum from the state of Rondônia. The compound 3286938 obtained an IC₅₀ of 24.4 µM against the W2 strain of P. falciparum, and against the circulating strains, it presented a median (MD)=38.7 µM for P. vivax and MD=6.7 µM for P. falciparum. As for toxicity, 3286938 showed CC₅₀ > 500 µM for VERO and HepG2 strains with a selectivity index greater than 12.9, a ratio calculated for P. falciparum and P. vivax regarding Vero and HepG2 cells. The compound was not considered hemolytic in in vitro assays, thus indicating the specificity of its antiplasmodial action. Based on the results presented, and considering the unprecedented character of the compound, it can be concluded that 3286938 was shown to be promising for complementary in vitro and in vivo studies aiming to produce effective antiplasmodial action.

Keywords:
Malaria; P. falciparum ; P. vivax ; Trimethoxy-phenyl

INTRODUCTION

Malaria is considered a serious disease by the World Health Organization (WHO) and it continues to have a major impact on public health, especially in tropical countries. According to WHO, it is estimated that more than one billion cases of malaria occurred worldwide between 2010 and 2018 (WHO, 2020). In Brazil, more than 99% of cases are concentrated in the Legal Amazon Region, due to its tropical climate and socioeconomic conditions which favor the proliferation, growth and survival of the anopheline vector (Lapouble, Santelli, Muniz-Junqueira, 2015; SINAN, 2019). Three species are associated with human infection in the country: Plasmodium falciparum, P. vivax and P. malariae. P. vivax is most predominant, followed by the species P. falciparum (Brazil, 2015).

Malaria caused by P. falciparum is considered the most serious form of the disease, leading to high mortality rates, with the potential to manifest neurological disorders, respiratory problems and may predispose women to the threat of miscarriage (Mackintosh, Beeson, Marsh, 2004). However, in recent years, P. vivax has been identified as the main cause of morbidity and deaths in babies and children in Southeast Asia (Kaushik, Gomber, Dewan, 2012; Singh et al., 2013). In addition, the clinical presentation of malaria caused by these two species can be aggravated due to the existence of isolates with remarkable genetic variation that are resistant to the antimalarials currently used, such as chloroquine (CQ) for P. vivax and artemisinin for P. falciparum; this becomes a threat to the control and elimination of the disease (Dondorp et al., 2009; Miotto et al., 2013).

The difficulty of controlling the disease and the emergence of strains resistant to current antimalarials makes the search for new drugs important to complement or replace the drugs currently used to treat malaria (Phyo et al., 2012; Ashley et al., 2014; Imwong et al., 2017).

In this context, we can highlight plants of the genus Piper L., Piperaceae which have components such as: amides, alkaloids, derivatives of benzoic acid, lignans, flavonoids and cinnamic acid (Facundo et al., 2008; Regasini et al., 2009). There are reports in the literature of trypanocidal, leishmanicidal and anti-inflammatory bioactivity for this genus (Calderon et al., 2009; Santos et al., 2018; Oliveira et al., 2018). In addition, there are other studies and reviews published in the literature demonstrating specific action of molecules derived from cinnamic acid (Guzman, 2014). These actions include antimalarial (Kanaani, Ginsburg, 1992), antifungal (Tawata et al., 1996), antioxidant, antimicrobial (Sova, 2012) and anticancer activities (Pontiki et al., 2014).

Many phenylpropanoids are derived from cinnamic acid, through reduction, hydroxylation and methylation reactions (Dewick, 2009). Studies and reviews published in the literature have demonstrated specific action of molecules derived from cinnamic acid (Guzman, 2014). The compounds that make up this family of metabolites were extracted, modified and synthesized for future studies. Santos et al. (2018) demonstrated that synthetic compounds derived from trimethoxyphenylpropanoic acid showed antiprotozoal action against Leishmania spp. and T. cruzi; study conducted by Ferreira et al. (2010) also reinforced the 3,4,5-trimethoxyphenyl group’s activity.

Therefore, due to the biological characteristics described above, this group can be an alternative in the search for new synthetics that can be used in the treatment of malaria. The compound 3286938 (Figure 1) presents in its structure the 3,4,5-trimethoxyphenyl group. Therefore, it was chosen for study due to its structural similarities with phenylpropanoid derivatives.

FIGURE 1
11-(3-benzyloxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-propan-1-one (CID: 3286938).

In this article, we report the activity of the synthetic compound 1-(3-benzyloxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-propan-1-one), a derivative of 3,4,5-trimethoxyphenyl propanoic acid, against P. falciparum strains in vitro and against P. vivax and P. falciparum strains circulating in the state of Rondônia.

MATERIAL AND METHODS

Ethical approval

This study was approved by the Research Ethics Committee of the Oswaldo Cruz Foundation - Rondônia, according to protocol numbers CAAE: 44899715.2.0000.0011 and CAAE 58738416.1.0000.0011.

Obtaining the compound

3286938 was obtained commercially by the company Sigma-Aldrich; its solubilization was carried out in dimethylsulfoxide (DMSO-Sigma-Aldrich) at a final concentration of 0.5%. Solubilization of the compound occurred on the day of the respective experiments.

In vitro assay against W2 P. falciparum

W2 P. falciparum parasites were grown in human erythrocytes according to criteria adapted from Trager, Jensen (1976). With the predominance of young trophozoites, the culture was synchronized using 5% D-sorbitol (Sigma-Aldrich) and the hematocrit was adjusted to 5%, according to conditions described by Lambros, Vanderberg (1979). The compound was tested in serial concentrations of 1000-1.5 µM. As a control of parasite death, artemisinin was used in serial concentrations of 0.17-0.001 µM, dihydroartemisinin (0.5-0.0002 µM). Infected red blood cells were used as a positive control and uninfected red blood cells as a negative control. The plates were maintained in an incubator at 37 °C for 48 hours (in a standard gas mixture consisting of 5% O₂, 5% CO₂ and 90% N₂) and stained using Sybr Green I (Invitrogen), which is a DNA intercalator. It is known that erythrocytes do not have nucleic acids, while parasites of Plasmodium spp. contain both DNA and RNA, so when parasites grow, there is also an increase in DNA capable of intercalating with Sybr Green I, resulting in increased fluorescence as described by Smilkstein et al. (2004).

Cytotoxicity Assay

The cytotoxic effect of the compound was evaluated against adherent HepG2 cells (derived from a human hepatoma) and VERO cells (kidney cells of the African monkey), using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide - Sigma-Aldrich) colorimetric assay, as described by Mosmann (1983). Cultivation took place in RPMI-1640 medium (Gibco) supplemented with 10% Bovine Fetal Serum (Gibco), followed by incubation at 37 °C with 5% CO₂, at 95% humidity (Calvo-Calle et al., 1994). For the toxicity tests, the cell suspension was adjusted to a proportion of 1x10⁴ cells/well in microplates and incubated again for 12 hours in order to achieve cell adhesion to the wells. After this incubation time, the cells were treated for 72 hours in serial concentrations from 500-3.9 µM for 3286938, artemisinin and dihydroartemisinin. Absorbance readings were obtained using a spectrophotometer (Biochrom Asys, Expert Plus, Holliston, USA) at 570 nm wavelengths. Untreated cells were used as the positive control and cells treated with 0.05% saponin (Sigma-Aldrich) were used as the negative control. The concentration cytotoxic to 50% of viable cells (CC₅₀) was calculated compared to cells with no treatment.

Hemolytic Assay

The hemolytic assay was performed with a suspension of 180 µL of human erythrocytes (1%) distributed in a 96-well microplate with a “U” bottom and subjected to treatment with the compound at serial concentrations (1000-1.5 µM). As a positive control of hemolysis, 0.05% saponin was used, while 1% red blood cells in incomplete RPMI-1640 medium were used as the negative control and RPMI-1640 alone was used as background. The plates were incubated in an incubator at 37°C for 30 minutes with shaking every 5 minutes (Wang et al., 2010). Then, the absorbance was read on a spectrophotometer at 540 nm wavelengths.

Ex vivo testing

Blood samples from patients infected with P. falciparum and P. vivax, with parasitemia between 11-100 parasites per 100 oil-immersion thick film fields, were collected between June 2017 and October 2018 at the Tropical Medicine Research Center (CEPEM) in the city of Porto Velho, state of Rondônia. Underage patients, indigenous people, pregnant women, people with mental disabilities and comorbidities, and patients who had used any antimalarial drug in the previous month or presented severe malaria were excluded from the study.

For ex vivo chemosensitivity assays, plates were prepared with the drug dihydroartemisinin tested at serial dilutions of 1-0.00024 µM and 3286938 at concentrations of 1000-1.5 µM; for both tests, the dilutions were done at proportions of 1:4. Initial parasitemia was verified using thick smear slides (stained using Giemsa). The white blood cells were removed by centrifugation, and the red cells were filtered through a CF11 cellulose column (Moody, 2002). The parasite culture was supplemented with McCoy medium for P. vivax and RPMI-1640 for P. falciparum (Marfurt et al., 2011). Parasite maturation was monitored after intervals of 24 and 48 hours of incubation, using thick blood films, from the untreated control. Incubation with antimalarials and compound 3286938 was stopped when at least 40% of schizonts were reached in 200 forms of parasites; the slide count was performed using optical microscopy.

Statistical analysis

The inhibitory concentration for 50% of parasites (IC₅₀) and the cytotoxicity concentration for 50% of cells (CC₅₀) were determined using dose-response curves based on non-linear regression; the curves were plotted using the program OriginPro. The selectivity index (SI) was obtained by calculating the ratio between CC₅₀/IC₅₀, where a value >10 is considered selective, and a value <10 is considered non-selective to parasites (Nava-Zuazo et al., 2010). Hemolytic activity was analyzed in GraphPad Prism and validated by analysis of variance (ANOVA), followed by Tukey’s post-test with a significance of p ˂ 0.05. In the descriptive analysis of the circulating strains, the measure of central tendency MD = median was used, being determined from the N sample of patients and calculating the interquartile interval and then plotted on GraphPad Prism.

RESULTS

Analogue 3286938 was initially tested in vitro against the W2 strain of P. falciparum. The IC₅₀ value obtained for this test was 24.4 µM (±1.4). The IC₅₀ value for the reference drugs (Figure 1 and Table I) used as a control showed results of 0.00026 µM (± 0.0) for artemisinin (ART) and 0.00065 µM (± 0.0) for dihydroartemisinin (DHART). Against circulating strains, the median IC₅₀ for compound 3286938 was 38.7 µM (MD 59.4) for P. vivax and 6.7 µM (MD 4) for P. falciparum. The median IC₅₀ for DHART, the reference drug used in the ex vivo assay, was 0.0011 µM (MD 0.001) against P. falciparum and 0.0051 µM (MD 0.003) against P. vivax (Figure 2 and Table I). The reference drug, artemisinin, was not tested against circulating strains.

FIGURE 2
Evaluation of in vitro and ex vivo activity of 1-(3-benzyloxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-propan-1-one) against Plasmodium falciparum (W2) and Plasmodium vivax. Note: 3286938= 1-(3-benzyloxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-propan-1-one); ART= artemisinin, reference drug used in the treatment of malaria; DHART= dihydroartemisinin, reference drug used in the treatment of malaria; P. falciparum (W2)= adapted laboratory strain; P. vivax= P. vivax strains isolated from patients; P. falciparum= P. falciparum strains isolated from patients. The IC₅₀ values for in vitro assays were obtained through the means and standard deviations of three independent experiments. The IC₅₀ values for the ex vivo tests with 3286938 and DHART were obtained using the median with a sample number of 10 patients for P. vivax and 6 patients for P. falciparum.

TABLE I
In vitro antimalarial activity

Compound	W2 Strains	Wild Strains			Toxicity			Selectivity index
	IC50 (µM) (±)	IC50 (µM) (MD quartile)			CC50 (µM) (±)			HepG2			VERO
	P. falciparum	P. falciparum	P. vivax		HepG2	VERO		SIa	SIb	SIc	SIa	SIb	SIc
3286938	24.4 ± 1.4	6.7 MD 4	38.7 MD 59.4		> 500	> 500		> 20.5	> 74.6	> 12.9	> 20.5	> 74.6	> 12.9
ART	0.00026 ± 0.0	NT	NT		> 500	> 500		>1.9	NC	NC	>1.923	NC	NC
DHART	0.00065 ± 0.0	0.0011 MD 0.001	0.0051 MD 0.003		32.05 ± 5	170.35 ± 1.34		49.3	29.1	6.284	26.2	154.9	33.4

• IC₅₀, CC₅₀ and SI values for the compound 1-(3-benzyloxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-propan-1-one) and reference drugs against Plasmodium falciparum (W2) and circulating strains of Plasmodium vivax and Plasmodium falciparum.
• Note: IC₅₀= Inhibitory Concentration of 50% of the parasites; CC₅₀= Cytotoxic concentration for 50% of the cell population. SI= Selectivity index (ratio between CC₅₀/IC₅₀); = average of IC₅₀ and CC₅₀; NT= not tested; NC= not calculated; quartile= median of IC₅₀ of circulating P. falciparum and P. vivax strains and interquartile range; ± standard deviation of the average of three experiments. 3286938= 1-(3-benzyloxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-propan-1-one), ART= artemisinin, DHART= dihydroartemisinin. SI^a= Selectivity index calculated for P. falciparum (W2). SI^b= Selectivity index calculated for circulating strains of P. falciparum. SI^c= Selectivity index calculated for circulating strains of P. vivax.

The compound 3286938 demonstrated an absence of toxicity at concentrations below 500 µM against HepG2 and VERO cell lines; the same result was observed for ART, with 100% cell viability at a concentration of 500 µM. For the reference drug DHART, the CC₅₀ value for HepG2 was 32.05 (± 5) and for VERO it was 170.35 (± 1.34) (Table I).

According to Table I, the selectivity index (SI) of the compound 3286938 was greater than 20.5 for both cells against the W2 strain in in vitro assays. For circulating strains of P. falciparum, the SI value was > 74.6.6 for HepG2 and VERO cells, and against P. vivax the SI value was >12.9 for both strains. Artemisinin, used as a control drug, obtained an SI value >1.923 (HepG2, VERO); this drug has not been tested against circulating strains. For the reference drug, DHART, the SI in vitro was 49.307 against HepG2 and 26.207 against VERO; for circulating strains of P. falciparum the SI obtained was 29.136 (HepG2) and 154.9 (VERO), and for P. vivax, SI of 6.284 (HepG2) and 33.401 (VERO) were obtained. Compound 3286938 was not considered hemolytic at a concentration of 1000 µM, the highest concentration used for the hemolysis assay.

The circulating P. vivax isolates showed IC₅₀ values for compound 3286938 ranging from 5.9 µM to 97.43 µM; the reference drug DHART ranged from 0.00078 to 0.0069 µM and 100% of the patients were male with ages ranging from 21 to 57 years old. Among the P. falciparum isolates, there was a smaller number of infected patients, with variation in the IC₅₀ values for DHART from 0.00034 µM to 0.0023 µM. For the compound 3286938, IC₅₀ varied between 0.49 µM and 12.3 µM. Of six isolated patients, four were male and two were female, with ages ranging from 21 to 57 years old (Table II).

TABLE II
Profile of patients infected with P. falciparum and P. vivax with lower sensitivity of the compound 1-(3-benzyloxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-propan-1-one) and Dihydroartemisinin detected by the ex vivo assay isolated from patients seen at CEPEM/RO in the state of Rondônia, from June 2017 to October 2018

Plasmodium vivax
Patient ID	Gender/ age	Parasites/µL	(% of Trophozoites at 0h)	Mature schizonts (%)	Incubation time (h)	IC50 µM
						DHART	3286938
170612 (01)	M/35	3000	94	56	24	0.0055	24
170711 (01)	M/41	3000	93	40	24	0.00078	97.43
170717 (02)	M/24	3000	93	66	24	0.00551	50.84
180202 (01)	M/51	2958.4	98.6	78	24	0.0062	86.8
180221 (01)	M/45	3000	100	40	48	0.0048	86.3
180312 (01)	M/43	2966.7	98.9	40	24	0.0019	5.9
180312 (02)	M/21	3000	100	40	30	0.0022	81.8
180328 (01)	M/37	3000	100	40	32	0.0062	25.7
180424 (01)	M/36	3000	100	45	36	0.0069	25.8
181015 (01)	M/57	3000	100	58	40	0.0045	26.61
Plasmodium falciparum
170614 (01)	M/30	3000	100	40	24	0.00078	5.27
170629 (02)	F/57	3000	100	40	24	0.0023	12.3
180730 (01)	F/36	3000	100	51	24	0.0017	8.1
180802 (01)	M/19	3000	100	45	24	0.00034	8.5
180815 (01)	M/51	2996.4	99.9	57	24	0.00146	4.1
180912 (01)	M/21	3000	100	40	27	0.00058	0.49

• Note: ID= Code; IC₅₀= Inhibitory Concentration of 50% of the parasites; DHART= dihydroartemisinin; 3286938= 1-(3-benzyloxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-propan-1-one).

Upon comparison of the incubation time of the isolates from the two species, it is noted that the incubation time for P. falciparum was shorter than that of P. vivax, due to the fact that P. falciparum has an adapted culture that is well described in the literature (Trager, Jensen, 1976), while P. vivax, in addition to having few previous studies, is difficult to maintain in a continuous culture both in vitro and ex vivo (Rangel et al., 2018).

DISCUSSION

Antimalarial activity of phenylpropene derivatives is poorly described in the literature. This class was studied by Palaniswamy et al. (2010); the authors mention that the ethanolic extract of Trigonella foenum-graecum L. showed IC₅₀ values of 8.75 ± 0.35 µg/mL and 10.25 ± 0.35 µg/mL against chloroquine-sensitive and resistant strains, respectively. According to the authors, after a phytochemical screening, tannin-phenolic compounds were found in the ethanolic extract; these compounds may have contributed to the antiplasmodial activity of Trigonella foenum-graecum L. The majority of phenolic compounds, like 3286938, the target of this study, are derived from plants’ metabolic pathways of phenylpropanoids.

A complementary study on antimalarial activity developed by Sharma et al. (2011), with methoxylated phenylpropenes against Dd2 (CQ-Resistant) P. falciparum, describes promising Inhibitory Concentrations for two phenylpropene derivatives with IC₅₀ values ranging from 4 to 6.8 µM. The results described by these authors, as well as the results observed by our group, with the derivative 3286938 (IC₅₀ 24.4 µM) against the W2 (CQ-Resistant) strain of P. falciparum illustrate the importance of this class of molecules in the search for new antimalarial prototypes.

Results of ex vivo screening with 3286938 against isolates from the region such as P. falciparum (IC₅₀ of 6.7 µM) and P. vivax (IC₅₀ of 38.7 µM) reinforce that this synthetic compound is a candidate for structural analysis seeking to improve and diversify a series of new derivatives.

Regarding studies involving the movement of the methoxy group in research on antimalarial activity, Liu, Wilairat, Go (2001) shows the antiplasmodial activity of the compound 1-(2,3,4-trimethoxyphenil)-3-(3-quinolinyl)-2-propen-1-one, with an IC₅₀ value of 2 µM against the K1 Chloroquine-resistant strain of P. falciparum. Casertano et al. (2020), like the authors mentioned above, worked with the methoxy group, more specifically with trimethoxybenzene, inserting substituents in the benzyl side chain. The results demonstrate IC₅₀ values from 5.02 to 0.81 µM and SI ranging from 45 to 11.1 against P. falciparum (CQ-Resistant) and HMEC-1 cells (human microvascular endothelial cells). The molecule from the present study, 3286938 1-(3-benzyloxy-4-methoxy-phenyl)-3-(3,4,5-trimethoxy-phenyl)-propan-1-one), which contains trimethoxy benzene as one of its groups, did not present such low IC₅₀ values; however, the SI values for HepG2 and VERO cells were higher than 20.5 when antimalarial activity against the W2 strain was considered; an SI value of 74.6 was observed for circulating strains of P. falciparum and an SI of 12.9 for circulating strains of P. vivax.

Regarding the structure of the molecules evaluated in the study by Yadav et al. (2012) and the compound 3286938, one similarity is the presence of a 3,4,5-trimethoxyphenyl group as a radical of the main molecules from the cited studies. Yadav et al. (2012) worked with chalcone derivatives, where the methoxy groups were inserted in different positions. Notably, 1-(4-benzimidazol-1-yl-phenyl)-3-(2,4-dimethoxy-phenyl)-propen-1-one presented an IC₅₀ value of 1.1 μg/mL against the 3D7 (CQ-sensitive) strain of P. falciparum. The authors concluded that the presence of two methoxy groups in positions 2 and 4 was excellent for antimalarial activity; in the sequence they had 3.4 dimethoxy and 2.5 dimethoxy with moderate activity. However, the 3,4,5-trimethoxy segment showed weaker activity, probably because it caused steric impediment in binding to the active site of the cysteine protease enzyme studied. This information indicates the possibility of more precise future investigations regarding structure-activity relationship (SAR) studies on the molecule 3286938, which presents the trimethoxy group in the same position as the study by Yadav et al. (2012).

When analyzing the results of the ex vivo antiplasmodial assays, it was observed that the circulating strains of P. falciparum and P. vivax had an IC₅₀ within expectations for the region in relation to DHART, with sensitivity for both (Aguiar et al., 2014). However, the IC₅₀ of the circulating strain of P. falciparum showed 4x less inhibition of parasitic growth than the W2 strain of the same species. It is noted that the W2 strain has been adapted to culture for years (Oduola et al., 1998), which is why these plasmodia naturally survive better in the mimetic conditions of the culture media and supplements, unlike the newly isolated strain. Another factor that influences the discrepancy between the IC₅₀ values is the fact that the antiplasmodial assay using the Sybr Green I technique (Smilkstein et al., 2004) takes into account parasitic growth as a function of the mass of DNA produced in the schizogony process was performed with W2 which lasted for 48 hours, while the schizont maturation test was performed in 24-27 hours with different parasitemias.

The literature points to the importance of the search for new antimalarial molecules due to the development of resistant strains. In this respect, P. vivax stands out for, despite few cases, already showing a loss of sensitivity to antimalarials in Brazil and worldwide (Brega et al., 2005; Suwanarusk et al., 2007). In this study, we observed that despite having a median of 38 µM against P. vivax, this was a pioneer study carried out with this class of molecules in the Amazon region as well as in the state of Rondônia. With attention increasing to finding new compounds with antimalarial effects, effective and low-cost synthetic molecules have become the target of antimalarial chemotherapy. The synthesis of derivatives of phenylpropanoids and substances that present the 3,4,5-trimethoxyphenyl group follows this pattern. The promising results obtained in this study demonstrate the potential of these substances in the development of new antimalarials.

Thus, we conclude that the molecule in question, 3286938, is a potential prototype for chemical improvement aiming to further increase the antiplasmodial activity against the two Plasmodium species tested and improve the cytotoxicity score and thus succeed in the in vivo and mechanism of action tests.

CONCLUSIONS

The results obtained with compound 3286938 showed antimalarial activity against W2 P. falciparum in vitro tests and ex vivo against circulating strains of P. falciparum and P. vivax in Porto Velho, Rondônia. In addition, it is possible to observe this molecule’s absence of cytotoxicity against HepG2 and VERO cells, which demonstrates possible specificity of this compound, highlighting it as an interesting prototype for further in vitro and in vivo investigations against protozoa that cause malaria.

ACKNOWLEDGMENTS

The authors would like to thank the Centro de Integração Empresa Escola in Porto Velho-Rondônia, Fundação Rondônia de Amparo ao Desenvolvimento das Ações Científicas e Tecnológicas e à Pesquisa do Estado de Rondônia (FAPERO) under Concession [number 0113100051-0000.84/2015], Instituto Nacional de Epidemiologia da Amazônia Ocidental-EpiAmO under Concession [number INCT-MCTI/ CNPQ/ CAPES/FAPs 16/2014], Programa Pesquisa para o SUS (PPSUS), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Programa Pró-Amazônia, and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial assistance during the execution of this study. The authors express their gratitude to Amy Grabner for the English review of this manuscript.

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Publication Dates

• Publication in this collection
  23 Jan 2023
• Date of issue
  2022

History

• Received
  09 June 2020
• Accepted
  16 Feb 2021