Chemicals used were: primaquine (Sigma, St Louis, MO, USA, #160393), paroxetine (Sigma, #P9623), nicotinamide adenine dinucleotide phosphate, oxidized form (NADP) (Sigma, # 077 K7000), acetonitrile (Fisher Scientific, Waltham, MA, USA, #972970), glucose-6-phosphate (G6P) (Sigma, # 046 K3779), glucose-6-phosphate dehydrogenase (G6PD) (Sigma, # 068 K3795), and magnesium chloride (MgCl2) (Sigma, #102 K0154). Mobile phases were made with HPLC grade water, acetonitrile and formic acid.
In vitro metabolism studies with the CYP2D6 isoenzyme were conducted according to the manufacturer’s instructions (BD Gentest, San Jose, CA, USA). Briefly, the procedure was as follows: a 30 μl aliquot of 5 mg/ml CYP2D6 was mixed with the NADPH regeneration system A (50 μl) and B (10 μl), and 990 ml of phosphate buffer (pH 7.4, 100 mM) was added. The solution was mixed gently by pipetting and incubated at 37°C for 2 min. Primaquine (10 μM final concentration) was added in the absence or presence of various concentrations of the CYP2D6 inhibitor paroxetine (0, 5, 10, 15, 50 μM). A portion of the mixture (120 μl) was then collected at several time points (0, 60 min) followed by quenching with an equal volume of acetonitrile. The samples were vortexed for 30 sec, and centrifuged at 13,200 × rpm at 4°C for 10 min. The supernatant was collected and loaded onto 96-well plates (200 μl/well) for LC-MS analysis.
Primaquine metabolite identification
Primaquine samples were analysed using a Waters (Milford, MA, USA) Acquity UPLC system coupled to a Xevo Q-ToF mass spectrometer equipped with a standard electrospray ionization source. Chromatographic separations were achieved using a Waters Acquity BEH C18 1.7 μm 2.1 mm × 100 mm column with a 2 to 98% acetonitrile gradient over 6.10 min at a flow rate of 0.70 mL/min. Mobile phase A consisted of 10 mM ammonium bicarbonate and mobile phase B consisted of acetonitrile. The gradient consisted of phase B increasing from 2 to 60% in the time period of 0 to 2.9 min, followed by 60 to 98% from 2.9 to 4.7 min, holding at 98% B from 4.7 to 5.2 min, and then returning to 2% B from 5.2 to 6.1 min. MS conditions were optimized for primaquine detection in the positive electrospray mode with the corresponding instrumental parameters: capillary 1 kV, sampling cone 20 V, extraction cone 4 V, source temperature 120°C, desolvation temperature 150°C, cone gas flow 30 L/Hr, and desolvation gas flow 600 L/Hr. Low energy MS scans were conducted using a collision energy of 6 V. Primaquine fragments were produced using the MSE mode with a collision energy ramp from 15–18 V. Primaquine metabolites were indentified and analysed using Waters Metabolynx software, MSE and MS/MS analysis.
IVIS study for C57BL/6 and knockout mice
PQ was administered orally on days −1, 0, and 1 with respect to sporozoite inoculation. At 24, 48, and 72 hours post-sporozoite infection, all inoculated mice were tested using the Caliper Life Sciences (Hopkinton, MA, USA) IVIS Spectrum instrument. Additionally, emerging blood stage infections were measured by a flow cytometry system (FC500 MPL, Beckman Coulter, Miami, FL, USA). Positive and negative controls are routinely used for the IVIS calibration in each test.
Sporozoites, inoculation and viability check
Plasmodium berghei sporozoites (luciferase expressing) were obtained from laboratory-reared female Anopheles stephensi mosquitoes from Department of Mosquito Biology, WRAIR and maintained at 18°C for 17 to 22 days after feeding on malaria infected Swiss CD-1/ICR mice. Salivary glands were extracted from malaria-infected mosquitoes and sporozoites were recovered by using in house procedures. Briefly, mosquitoes were separated into abdomen and head/thorax. Heads and thoraxes were triturated with a mortar and pestle and suspended in medium RPMI 1640 containing 1% C57BL/6 mouse serum (Rockland Co., Gilbertsville, PA, USA). A total of 50–80 heads with glands were placed into a 0.5 ml Osaki tube on top of glass wool with enough dissection media to cover the heads. The Osaki tube was kept on ice until all mosquitoes had been dissected. Sporozoites isolated from the same batch of mosquitoes were inoculated into C57BL/6, 2DKO and 2DKO/KI C57BL/6 mice on the same day to control for biological variability in sporozoite preparations. Each mouse was inoculated intravenously in the tail vein with approximately 10,000 sporozoites suspended in 0.1 ml volume on day 0.
To ensure that inoculated sporozoites were viable following the isolation procedure, they were stained with a vital dye containing fluorescein diacetate (50 mg/ml in acetone) and ethidium bromide (20 μg/ml in phosphate-buffered saline; Sigma Chemical Co., St Louis, MO, USA) and counted in a haemocytometer. The viability of sporozoites ranged from 90 to 100%.
Male eight-week-old C57BL/6, 2DKO and 2DKO/KI mice (Taconic, Hudson, NY, USA) were used. On arrival, the animals were acclimated for seven days (quarantine). The animals were housed in a cage maintained in a room with a temperature range of 18-26°C [express in °C], 34-68% relative humidity and a 12-hr light/dark cycles. Food and water were provided ad lib during quarantine and throughout the study. The animals were fed a standard rodent maintenance diet. All animal studies were performed under IACUC approved protocols. All animal use, care and handling were performed in accordance with the current Guide for the Care and Use of Laboratory Animals (1996).
Test agents and administration
The compounds tested in these experiments were dosed based on the body weight at the time of preparation of the suspension solution. The suspension solution of oral agents were prepared in 0.5% (w/v) hydroxyethyl cellulose and 0.2% (0.5% HECT, v/v) Tween-80 in distilled water, using homogenizer (PRO Scientific Inc, Monroe, CT, USA) with 10 mm open slotted generator to homogenize drug powder mixture at 20,000-22,000 rpm for 5 min in ice bath. A once-a-day, three-consecutive-day treatment regimen (−1, 0, 1 day) was used in all assessments. This volume was transferred to a 20-ml bottle, drawn into a 1-ml syringe, and delivered via intragastric feeder (18-gauge) to the designated recipient.
In vivo imaging of luciferase activity from luciferase expressing P. berghei-infected mice was performed using a Xenogen IVIS-200 Spectrum (Caliper Life Sciences, Hopkinton, MA, USA) in vivo imaging system. Mice were evaluated at 24, 48 and 72 hours post-sporozoite inoculation to determine liver- and blood-stage malaria infection. D-Luciferin potassium salt, (Xenogen, California and Goldbio, St Louis, MO, USA), the luciferase substrate, was intraperitoneally inoculated into mice at a concentration of 200 mg/kg 15 min before bioluminescence analysis. The mice were anaesthetized with isoflurane 3 min post-luciferin administration. The mice were then positioned ventral side up in the IVIS on the 37°C platform. The mice continued to receive isoflurane through the nose-cone delivery. The camera exposure time was 5 min for the 24, 48 and 72-hr time points with f-stop = 1 and large binning setting. Photons emitted from specific regions were quantified using Living Image® 3.0 software and total ROI was calculated. 3-D bioluminescent imaging tomography was performed with Living Image 3.0 software using sequential images taken with filters ranging from 580 to 660 nm.