Antiplasmodial activity, in vivo pharmacokinetics and anti-malarial efficacy evaluation of hydroxypyridinone hybrids in a mouse model
© Dambuza et al. 2015
Received: 15 July 2015
Accepted: 2 December 2015
Published: 16 December 2015
During the erythrocytic stage in humans, malaria parasites digest haemoglobin of the host cell, and the toxic haem moiety crystallizes into haemozoin. Chloroquine acts by forming toxic complexes with haem molecules and interfering with their crystallization. In chloroquine-resistant strains, the drug is excluded from the site of action, which causes the parasites to accumulate less chloroquine in their acid food vacuoles than chloroquine-sensitive parasites. 3-Hydroxylpyridin-4-ones are known to chelate iron; hydroxypyridone-chloroquine (HPO-CQ) hybrids were synthesized in order to determine whether they can inhibit parasites proliferation in the parasitic digestive vacuole by withholding iron from plasmodial parasite metabolic pathway.
Two HPO-CQ hybrids were tested against Plasmodium falciparum chloroquine-sensitive (D10 and 3D7) and -resistant strains (Dd2 and K1). The pharmacokinetic properties of active compounds were determined using a mouse model and blood samples were collected at different time intervals and analysed using LC–MS/MS. For in vivo efficacy the mice were infected with Plasmodium berghei in a 4-day Peters’ test. The parasitaemia was determined from day 3 and the course of the infection was followed by microscopic examination of stained blood films every 2–3 days until a rise in parasitaemia was observed in all test subjects.
IC50 values of the two compounds for sensitive and resistant strains were 0.064 and 0.047 µM (compound 1), 0.041 and 0.122 µM (compound 2) and 0.505 and 0.463 µM (compound 1), 0.089 and 0.076 µM (compound 2), respectively. Pharmacokinetic evaluation of these compounds showed low oral bioavailability and this affected in vivo efficacy when compounds were dosed orally. However, when dosed intravenously compound 1 showed a clearance rate of 28 ml/min/kg, an apparent volume of distribution of 20 l/kg and a half-life of 4.3 h. A reduction in parasitaemia was observed when compound 1 was dosed intravenously for four consecutive days in P. berghei-infected mice. However, a rise in parasitaemia levels was observed on day 6 and on day 9 for chloroquine-treated mice.
The hybrid compounds that were tested were able to reduce parasitaemia levels in P. berghei-infected mice when dosed intravenously, but parasites recrudesced 24 h after the administration of the least dose. Despite low oral bioavailability, the IV data obtained suggests that further structural modifications may lead to the identification of more HPO-CQ hybrids with improved pharmacokinetic properties and in vivo efficacy.
KeywordsMalaria Pharmacokinetics Hydroxypyridone-chloroquine In vivo efficacy
Malaria is a disease caused by an eukaryote parasite of the genus Plasmodium and remains a major global public health problem, that was responsible for 584,000 deaths in 2013, with most occurring in Africa and most deaths in children under 5 years of age [1, 2]. Plasmodium falciparum and Plasmodium vivax, are responsible for most cases of malaria and the control of these parasites by chloroquine and other known anti-malarial drugs has been compromised by the emergence and spread of drug resistance in many parts of the world, primarily in P. falciparum strains [3, 4]. This has severely limited the use of many effective anti-malarials, and has become a major threat to malaria elimination efforts, causing increased morbidity and mortality and a financial burden due to sustenance of replacement therapy .
When the parasite infects the erythrocyte, it uses the endolysosomal system to digest the haemoglobin in an acidic food vacuole, producing an oxidized form of haem, ferriprotoporphyrin IX (FP-IX), which is the iron containing non-protein component of haemoglobin, as a by-product [5–7]. Free FP-IX is toxic and can lyse the cell and affect the function of lysozomal enzymes. The parasite disposes the toxic FP-IX by a polymerization process that crystallizes at least 95 % of FP-IX as haemozoin, and this allows an uninterrupted growth and proliferation of the parasite [8–10]. Iron chelators have been studied as alternative malaria drugs for many years because of their ability to interact with available iron in the nucleus and parasite cytosol, thereby interfering with the iron-dependent metabolism of malaria parasites and inhibiting their development [11, 12].
Hydroxypyridones are iron-chelating agents known to suppress malaria growth in vivo and in vitro [13, 14]. Both hydroxypyridones and chloroquine target the erythrocytic stage of the malaria life cycle, which is highly dependent on iron. For the purpose of this study, N-alkyl-3-hydroxypyridin-4-ones were combined with chloroquine in an attempt to enhance antiplasmodial effect against chloroquine-resistant strains of P. falciparum when compared to inhibition by chloroquine alone . Compound 1 and 2 were selected among a series of hydroxypyridone-chloroquine (HPO-CQ) hybrid compounds in order to assess their antiplasmodial activity in vitro and efficacy in vivo and to evaluate their pharmacokinetic properties.
In vitro antiplasmodial activity against chloroquine-sensitive and resistant strains
The human parasite P. falciparum strains used in this study were chloroquine-sensitive (CQS) (3D7 and D10) and chloroquine-resistant (CQR) (Dd2 and K1) and were obtained from the malaria reagent depository, Malaria Research and Reference Reagent Resource Center (MR4) (ATCC, Manassas, VA, USA). The parasites were continuously cultured in vitro according to the method described by Trager and Jensen but with modifications . The cultures were maintained at 37 °C in O-positive (O+) human erythrocytes in complete culture medium, which contained RPMI 1640 (Gibco-BRL Laboratories) growth medium supplemented with 25 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) (Sigma-Aldrich Chemical Company) buffer, 22 mM glucose (Sigma), 5 g/l albumax (Gibco-BRL Laboratories), 25 mM sodium carbonate (Sigma) and 0.3 mM hypoxanthine (Sigma). In order to control microbial contamination 50 µg/l gentamicin (Sigma) was added to the culture medium, and to obtain a loosely synchronous ring stage, 5 % sterile aqueous d-sorbitol (Sigma) was used. The antiplasmodial assay was initiated with the parasites in the trophozoite stage and with a parasitaemia and haematocrit of 2 %.
The test compounds were tested at a concentration range of 0.15–150 mM and chloroquine (Sigma) was tested at a concentration range of 0.003–3 and 0.0003–0.3 mM for resistant strain and sensitive strains, respectively. Non-parasitized erythrocytes were used as a negative control and parasitized erythrocytes, without any test compound, were used as positive control. A full day dose–response was performed for all compounds to determine the concentration inhibiting 50 % of parasite growth (IC50 value). The plates were incubated for 48 h at 37 °C in a gassing chamber containing a mixture of 5 % CO2, 3 % O2 and 92 % N3. In order to quantify parasite viability and to determine the effect of the compounds on the parasite, a parasite lactate dehydrogenase (pLDH) assay was used as described by Makler et al. [16, 17], but with modifications.
A comprehensive pharmacokinetic (PK) study of compound 1 and 2 was performed on 10-week old male and female C57BL/6 mice (20–30 g) obtained from the University of Cape Town Medical School Animal Unit. The mice were housed in ventilated cages at room temperature (approximately 22 °C) with constant supply of food and water and were monitored twice daily. The study was authorized by the Faculty of Health Science Animal Research Ethics Committee before commencement: Reference No. 012/020. All the work was performed according to the guidelines established by Austin et al. .
The compounds were prepared in oil-in-water (o/w) microemulsions, which consist of 5 % ethyl linoleate (Sigma), 11 % Tween 80 (Sigma), 4 % ethanol (Merck), and 80 % water. In each experiment a group of five animals were dosed orally (10 ml/kg) at 20 mg/kg and intravenously (5 ml/kg) via the dorsal penile vein at 4 mg/kg under anaesthesia. For oral dosage, a gavage needle was used for the administration of test compounds directly into the lower oesophagus or stomach. Blood samples (approximately 30 µl) were collected serially by needle prick on the tail vein, near to the tip of the tail, at 0, 0.17, 0.5, 1, 2, 3, 5, 7, and 9 h post-dosing. Lithium heparin-coated MiniCollect® Plasma Tubes (Lasec, South Africa) were used to collect the blood samples. The collected blood samples were placed on ice immediately after sampling, and were frozen at −80 °C until analysis.
The blood samples stored at −80 °C were thawed at room temperature and then mixed by vortex to ensure homogeneity. Twenty µl of blood was mixed with 50 µl Milli-Q water (Millipore, USA) and 150 µl acetonitrile (Merck). The mixture was vortexed for 15 s, sonicated for 10 min and centrifuged at 13,000g for 5 min. The supernatant was transferred to a flat-bottomed glass insert and placed in a glass vial and placed in the autosampler for analysis.
Liquid chromatography–mass spectrometry summary
A liquid chromatography–mass spectrometry (LC/MS/MS) system was employed for the quantification of the compounds in mouse blood. The LC system employed was an ultra-fast liquid chromatography (UFLC) system (Shimadzu) and the separation of the compounds was performed on a Phenomenex, Luna 5 μm PFP (2), 100 Å, 50 mm × 2 mm analytical column. The mobile phase A consisted of 0.1 % formic acid in water (v/v) and mobile phase B consisted of acetonitrile. The flow rate was set at 500 µl/min and the temperature of the column was maintained at 40 °C. For the separation of the compounds, the mobile phase was increased from 5 to 95 % B over 4 min, after that, phase B was returned to 5 % within 0.1 min, then equilibrated for 3 min.
Mass spectrometer settings and MS parameters used for the detection of the test compounds on an API 3200 Q-Trap
Q1 mass (Da)
Q3 mass (Da)
Dwell time (ms)
Declustering potential (V)
Collision energy (V)
Entrance potential (V)
Collision cell exit potential (V)
Source temperature (°C)
Curtain gas (psi)
Gas 1 (psi)
Gas 2 (psi)
Ion spray voltage (kV)
Non-compartmental analysis was performed on each individual set of data using PK Solutions 2.0 Pharmacokinetic Analysis Software (Summit Research Services, Montrose, USA). The following PK parameters were calculated: apparent terminal half-life [t½ (min)], total exposure [AUC0–α (µM min)], volume of distribution (l/kg) and plasma clearance [CL (l/min/kg)].
In vivo anti-malarial efficacy of compound 1 against a chloroquine-sensitive Plasmodium berghei strain
The chloroquine-sensitive Plasmodium berghei (ANKA strain) was used to assess in vivo anti-malarial efficacy of the test compounds. The parasites were maintained in a C57BL/6 mouse by inoculation with 250 µl of a 1:1 (v/v) suspension of erythrocytes infected with P. berghei in phosphate buffered saline (PBS). On the day of the experiment the host mouse was anaesthetized intraperitoneally with a mixture of ketamine (120 mg/kg) and xylazine (16 mg/kg). Whole blood from the host mouse was drawn by cardiac puncture into a Vacuette® heparin tube and a suspension of P. berghei parasitized erythrocytes (1 × 107) in PBS was prepared and the test mice were infected with 200 µl of this suspension intraperitoneally.
Antiplasmodial action of the compounds in vitro
In vitro IC50 values (µM) of compounds 1 and 2
0.064 ± 0.02
0.047 ± 0.01
0.505 ± 0.10
0.463 ± 0.12
0.041 ± 0.02
0.122 ± 0.03
0.089 ± 0.01
0.076 ± 0.01
0.019 ± 0.00
0.023 ± 0.00
0.279 ± 0.002
0.180 ± 0.01
Pharmacokinetic parameters of hydroxypyridone-chloroquine hybrids after intravenous administration
4.3 ± 1.02
0.7 ± 0.2
20.1 ± 6
154.1 ± 1
27.6 ± 8
366 ± 46.6
AUC0–∞ (µM min)
196 ± 29.9
25.2 ± 3.4
Compound 1 showed a clearance rate of 28 ml/min/kg, a high apparent volume of distribution of 20 l/kg and a half-life of 4.3 h. The area under the curve (AUC) value was also at 196 µM min. Even with the half-life of 4.3 h, the compound remained at detectable levels of 0.16 µM after 9 h at the end of the dosing interval, indicating a slow elimination rate from circulation. When considering these properties and IC50 values of 0.064–0.505 µM against sensitive and resistant strains, respectively, compound 1 was selected for in vivo efficacy studies. Even though the compound could not be detected in blood following an oral dosage, investigating the efficacy of compound 1 after an intravenous dosage was considered.
Compound 2, on the other hand, had a very short half life of 0.7 h and after 2 h the blood levels dropped to 0.03 µM because of a high clearance rate of 366 ml/min/kg. The volume of distribution was also high at 154.1 l/kg.
Anti-malarial effect of compound 1 on Plasmodium berghei-infected mice
When considering compound 1 blood concentration of healthy animals in Fig. 2a and P. berghei-infected animals in Fig. 3, it was noted that higher drug levels were observed in infected mice. It is unlikely that the presence of parasites could be the reason for such a significant difference in blood levels because the mouse exposure study was performed 2 h post infection and parasitized erythrocytes are rapidly absorbed after intraperitoneal inoculation . This difference could possibly be due to the variation in age and weight.
To evaluate the anti-malarial efficacy of compound 1 in infected mice, the parasitaemia level was calculated at days 4, 6 and 9 post infection, and the % reduction of parasitaemia was calculated for day 9 only because parasite levels were observed in all treated mice on day 9.
In vivo antiplasmodial efficacy of compounds 1 in C57BL/6 mice blood infected with P. berghei
Average % parasitemia
% reduction of parasitemia
Oral (20 mg/kg/day)
5.3 ± 3.9
22.7 ± 10
24.2 ± 5.6
3.7 ± 5.6
Oral (40 mg/kg/day)
5.9 ± 2
10.7 ± 5
48.6 ± 12
−93.8 ± 49.3
IV (4 mg/kg/day)
0.4 ± 0.4
1.8 ± 1.4
8.1 ± 5
61.9 ± 26
IV (8 mg/kg/day)
0.9 ± 0.9
5.1 ± 1.7
82.9 ± 2.5
Oral CQ (10 mg/kg/day)
2 ± 1.6
92.0 ± 0.4
2.5 ± 2.4
7.3 ± 2.4
25.1 ± 0.4
As expected, blood levels remained high for a longer period for mice dosed intravenously with 4 and 8 mg/kg of compound 1 as shown in the concentration versus time profiles in Fig. 3. These data are consistent with the comprehensive PK study presented in Table 3, which shows that compound 1 had a half-life of 4.3 h following intravenous dosage and a clearance rate of 28 ml/min/kg. Figure 3 shows that after 7 h following intravenous dosage the blood concentration levels were relatively high for 4 and 8 mg/kg. This resulted in a reduced parasite multiplication rate as indicated by undetected to low parasitaemia until day 4 and 6. However, the data suggest that compound 1 suppressed the parasitaemia without clearing all parasites, therefore, surviving parasites in the mice blood began to multiply after the 4-day course of treatment, causing a parasite reduction to decrease to 62 and 83 % in the mice dosed at 4 and 8 mg/kg on day 9, respectively, as shown in Table 4. This also shows that the efficacy of compound 1 is dose dependent.
The aim of synthesizing HPO-CQ hybrids was to develop anti-malarial candidates that were potent against sensitive and resistant P. falciparum strains. This study showed that combining chloroquine with a HPO can cause a change in the physicochemical properties of the hybrid, thus affecting its efficacy and PK properties. Chloroquine alone has an oral bioavailability of 79–90 % and a half-life of 7 h in mice, as reported by Salako . Even though compound 2 was more active than chloroquine in vitro against the resistant strains with a lower RI value, the PK properties were not improved relative to chloroquine. Compound 1 was less active in vitro against the resistant strains than compound 2 and chloroquine with poor oral bioavailability, but it proved to be an effective anti-malarial compound in vivo when dosed intravenously. Nevertheless, chloroquine remained more effective in vivo than both compounds and this means that further modifications on the chemical structure could improve PK properties, such as oral bioavailability, thus improving efficacy.
area under the curve
liquid chromatography–mass spectrometry
limit of quantitation
multiple reaction monitoring
phosphate buffered saline
red blood cells
- T1/2 :
ultra-fast liquid chromatography
volume of distribution
ND participated in research design, conducted in vitro and in vivo experiments, interpreted the data and wrote the manuscript. PS, KC and LW participated in research design and contributed to the writing of the manuscript. AE developed and validated the LC–MS/MS assay for the quantitative determination of compounds 1 and 2 in mouse blood. DT performed in vivo efficacy experiments. JN assisted with the evaluation of the PK-properties using PK-summit software. AA and TE synthesized the compounds. All authors read and approved the final manuscript.
The authors acknowledge Mr. Trevor Finch for assisting with animal handling. The University of Cape Town, Clinical Infectious Diseases Research Initiative and National Research Foundation, South African Medical Research Council, and South African Research Chairs Initiative of the Department of Science and Technology, administered through the South African National Research Foundation are gratefully acknowledged for support.
The authors declare that they have no competing interests.
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