Chemicals and reagents
Naphthoquine phosphate was purchased from Kunming Pharmaceutical Corporation (KPC, purity > 98.0%, Yunnan, China). Chloroquine (CQ) was purchased from the National Institutes for Food and Drug Control (purity > 99.0%, Beijing, China). Amodiaquine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals used were purchased from Sigma-Aldrich or Fisher Scientific.
Parasite strain
The murine malaria parasite Plasmodium yoelii was obtained from the Malaria Research and Reference Reagent Resource Center (MR4) as a part of the BEI Resources Repository, National Institute of Allergy and Infectious Diseases, National Institute of Health.
Animal handling
ICR mice (20–25 g) and Wistar rats (200–220 g) were supplied by the Laboratory Animal Centre of Shandong University (Grade II, Certificate No. SYXK2013-0001). The experimental protocol was approved by the University Ethics Committee and conformed to the “Principles of Laboratory Animal Care” (NIH publication no. 85-23, revised 1985). Laboratory animals were fasted for 12 h before drug administration and for a further 2 h after dosing. Water was freely available during experiments.
In vivo antiplasmodial activity
Male or female ICR mice were treated intraperitoneally (i.p.) with 1 × 107 red blood cells (RBCs) infected with P. yoelii. The positive model drug (CQ; dissolved in 0.03% acetic acid) was orally administered at 24, 48 and 72 h post-infection. To simulate the clinical use of NQ as a single-dose regimen, the tested drug NQ (dissolved in 0.03% acetic acid) was orally given at 24 h post-infection. Efficacy was carried out using five different dose levels with nine mice at each level. Parasitaemia was assessed by microscopic examination of Giemsa-stained blood smears on day 4 post-infection. The 50% or 90% growth inhibitory doses (ED50 or ED90, respectively) of NQ were an average of three independent measurements (three mice in each dose group). Mice without the parasitaemia were considered fully cured.
Pharmacokinetic study
Healthy male or female rats (n = 7 for each group) were given a single oral dose of NQ (40 mg/kg; dissolved in 0.03% acetic acid). Blood samples (150 μL) were withdrawn before dosing and at 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, 60, 72, 84, 96, 120, 144, 168, 240, 336, 504, 672 and 840 h after dosing. Heparinized blood samples were centrifuged and plasma samples were stored at − 20 °C until analysis.
Quantification of NQ
Plasma samples were subjected to a protein precipitation extraction process. In brief, 20 μL of rat plasma was mixed with 4 μL of 0.03% hydrochloride acid, followed by addition of 10 μL of internal standard (IS, amodiaquine, 200 ng/mL) and 200 μL of acetonitrile. The samples were mixed and centrifuged at 14,000 rpm for 20 min. The supernatant was evaporated to dryness at 45 °C in a speedVac concentrator, and the residue was reconstituted in 150 μL of the initial mobile phase before LC–MS/MS analysis. For calibration preparation, 20 μL of drug-free plasma was mixed with 4 μL of stock solution (prepared in 0.03% hydrochloride acid), 10 μL of IS and 200 μL of acetonitrile. The mixture was treated as above. Matrix-matched calibration standards were obtained with concentrations of 1.0–400.0 ng/mL for NQ. The analytical method was fully validated according to the Guidelines on bioanalytical method validation drafted by US Food and Drug Administration (2013), which included selectivity, linearity, accuracy and precision, matrix-effect, recovery, dilution integrity, carryover and stability.
An LC–MS method was applied for quantification of NQ on an API5500 Q-Trap triple quadrupole mass spectrometer (AB SCIEX, Concord, Ontario, Canada) equipped with a TurboIonSpray source. The chromatographic separation was achieved on a Poroshell 120 SB-C18 column (100 × 4.6 mm i.d., 2.7 μm, Agilent Technologies) at 40 °C. The mobile phase consisted of (A) acetonitrile and (B) 0.2% formic acid and 0.05% trifluoroacetic acid, delivered at a flow rate of 0.6 mL/min. The HPLC gradient system started with 30% A for 0.5 min and linearly increased to 90% A in 2.5 min, followed by a decrease to 30% A prior to column re-equilibration. The electrospray ion source was operated in the positive ionization mode. The ionization voltage was + 3.5 kV and the source temperature was set at 550 °C. Nitrogen was used as the curtain gas (40 psi), nebulizer gas (GS1, 55 psi) and turbo gas (GS2, 55 psi). The multiple reaction monitoring (MRM) transitions were m/z 410.0 → 337.1 and m/z 356.0 → 283.1 for NQ and IS, respectively.
Plasma protein binding
Plasma protein binding (PPB) of NQ (0.1 and 1.0 μg/mL) was determined in pooled plasma collected from female or male mice, rat or human, using the ultrafiltration method. Briefly, stock solution of NQ was diluted with blank plasma to achieve the test concentrations. Incubations were performed in a shaking water bath at 37 °C for 1 h to allow equilibration. Plasma samples were loaded into the Ultra centrifugal filters (Millipore, USA) with 10 kDa molecular weight cutoff, and the filtrate was centrifuged at 6000 rpm for 20 min at 37 °C. Phosphate buffered saline (PBS) was used to test non-specific binding (NSB). The NSB was calculated according to the equation:
$$ {\text{NSB}}\, = \,\left( {{\text{C}}_{\text{BD}} - {\text{C}}_{\text{BF}} } \right)/{\text{C}}_{\text{BD}} , $$
where CBD was the total drug concentration in PBS before centrifugation and CBF was the drug concentration in the PBS filtrate after centrifugation. The PPB was calculated based on the equation:
$$ {\text{PPB}}\% \, = \, 100\, \times \,\left( { 1 - {\text{C}}_{\text{SF}} /\left[ {\left( { 1 - {\text{NSB}}} \right)\, \times \,{\text{C}}_{\text{SD}} } \right]} \right), $$
where CSF was the NQ concentration in the plasma ultrafiltrate and CSD was the nominal plasma concentration. All drug concentrations were determined by LC–MS/MS.
Metabolic clearance of NQ in liver microsomes
Pooled liver microsomes derived from male or female mice, rat or human were purchased from RILD Research Institutes for Liver Diseases (Shanghai, China). NQ (10 μM) was incubated with pooled male or female liver microsomes derived from three species, i.e., mice (MLM), rat (RLM) and human (HLM) (1 mg/mL) in potassium phosphate buffer (0.1 M, pH 7.4) and NADPH (1 mM) at 37 °C for 1 h. The incubation was initiated by adding NADPH and stopped by adding two volumes of cold acetonitrile. After centrifugation at 3000g for 10 min, the supernatant was dried under N2 at 45 °C and then reconstituted with initial mobile phase. An aliquot of the reconstituted sample was analyzed by LC–MS/MS. The in vitro intrinsic clearance (CLint, in vitro) was calculated according to the equation:
$$ {\text{CL}}_{{{\text{int}},{\text{in}}\;{\text{vitro}}}} \, = \,\left( {0. 6 9 3/{\text{t}}_{ 1/ 2} } \right)\, \times \,\left( { 1/{\text{C}}_{\text{protein}} } \right), $$
where Cprotein was the protein concentration.
Data analysis
Drug susceptibility was analysed by a nonlinear regression of logarithmically transformed concentrations. The doses that inhibited parasite growth by 50% (ED50) and 90% (ED90) were determined for NQ against P. yoelii in infected male or female mice. The peak plasma concentration (Cmax) and the time to peak concentration (tmax) were obtained from experimental observations. The other pharmacokinetic parameters were analyzed by use of a non-compartmental model and the program TOPFIT (version 2.0; Thomae GmbH, Germany). The area under the plasma concentration–time curve (AUC0–t) was calculated using the linear trapezoidal rule to approximately the last point. Total oral body clearance (CL/F) was calculated as dose/AUC0–t. The terminal elimination half-life (t1/2) was estimated by log-linear regression in the terminal phase using an average of five observed concentrations.
Results were expressed as mean ± SD. Comparison of the pharmacokinetic parameters (AUC0–t and Cmax) were performed after logarithmic transformation, and the mean changes in pharmacokinetic parameters among different groups were compared using Student’s t-test, which were performed with SPSS (version 19.0, SPSS Inc., Chicago, IL, USA). The comparison of tmax for the different treatment groups was performed using the Wilcoxon signed-rank test. The acceptable level of significance was established at P < 0.05. A greater than 1.5 increase in AUC0–t or antiplasmodial activity (ED50 or ED90), relative to the control, was defined to be significant.