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Ivermectin metabolites reduce Anopheles survival
Metabolite production
Powdered ivermectin parent compound was obtained from Sigma-Aldrich (St. Louis, Missouri, USA). The M3 (4-hydroxymethyl ivermectin) metabolite was synthesized by synthetically modifying the ivermectin parent compound (WuXi AppTec (Tianjin) Co., Ltd, Tianjin, China). The M1 (3″-O-demethyl ivermectin) metabolite was generated by exposure of ivermectin parent compound to biotransformation by a proprietary bacteria strain (Hypha ID: Sp159) at Hypha Discovery. First, a screening process was performed wherein ivermectin parent compound was subjected to whole-cell biotransformations using different bacterial species and strains known to produce oxidized metabolites of exogenous compounds. The bacterial culture extracts were shipped to MORU for evaluation by LC–MS/MS to identify which strain produced only the target M1 structure. The LC–MS/MS system used was an ultra-high performance liquid chromatography (Agilent 1260 Quaternary pump, Agilent 1260 High Performance autosampler, and Agilent 1290 Thermostatted Column Compartment SL, Agilent Technologies) coupled to a quadrupole time-of-flight mass spectrometer (Q-TOF–MS) (TripleTOF 5600+, Sciex) with an electrospray ionization using a DuoSpray ion source9. Once the ideal bacterial strain for production of M1 was identified, then bacterial biotransformation was scaled up in one 2L batch exposed to 200 mg of ivermectin (i.e., 100 μg/mL) which, after extraction and purification, produced a final quantity of 41.8 mg of the M1 metabolite. No bacterial strains were identified which produced M6 (3″-O-demethyl, 4-hydroxymethyl ivermectin) in sufficient, tractable quantities directly from ivermectin. Therefore, an additional lot of M3 was synthesized and 200 mg was biotransformed using 2 × 1L batches of the same bacterial strain used to produce M1. This resulted in 6.0 mg of the extracted and purified M6 metabolite. Identities of the M1, M3 and M6 metabolite products were confirmed by near magnetic resonance (NMR) spectroscopy. Further information on these processes and structural elucidation aspects are provided in the Supplementary Materials.
Ethics approval and consent to participate
The study protocol was approved by the ethics committees of the Faculty of Tropical Medicine, Mahidol University (MAL18006), the Oxford University Tropical Research Ethics Committee (OXTREC 33-18), and the Walter Reed Army Institute of Research (WRAIR#2609). All experiments were performed in accordance with the relevant guidelines and regulations of the above listed institutions. Each blood donor volunteer was provided with an explanation of the study and signed a written informed consent before study entry.
Mosquito blood meal preparation
Whole blood was collected from healthy volunteers on the day of each mosquito membrane feed. Blood was drawn into sodium heparin tubes. Compounds were dissolved in dimethylsulfoxide (DMSO) to concentrations of 2 mg/mL and 12 μL aliquots were frozen at − 20 °C. Compounds were thawed and serial dilutions were made in phosphate buffered saline (PBS) with 10 μL added to 990 μL of blood to reach final concentration desired for mosquito membrane feeding assays. Control blood meals consisted of previously frozen DMSO diluted in PBS to match the highest ratio of DMSO and PBS fed to mosquitoes in the compound-containing blood meals. A 50 µL aliquot of each mosquito blood meal was frozen at − 80 °C for LC–MS/MS analysis.
Mosquitoes
All mosquitoes were reared at the Armed Forces Research Institute of Medical Sciences Department of Entomology in Bangkok, Thailand. Anopheles dirus s.s. and An. minimus s.s. were produced as described previously17. Adult mosquitoes used for experiments were provided 10%sucrose solution ad libitum. Mosquitoes were reared at 25 ± 2 °C and 80 ± 10% relative humidity, and a 12 h light:12 h dark photoperiod. Mosquitoes were between 5 and 7 days post emergence at time of blood feeding, and were sugar-starved with access to water from 12 to 18 h before their blood meal.
Mosquito membrane feeding and mortality assays
At each mosquito membrane feed 1 mL of whole blood mixed with the different compounds over a range of concentrations were offered to groups of 40 An. dirus and 40 An. minimus mosquitoes via membrane feeders warmed to 37 °C. After feeding, up to 30 blood-fed mosquitoes of each species were gently transferred via aspiration to clean cardboard containers (0.5 L). After the blood meal, mosquitoes were maintained in an incubator at 25 ± 1 °C and 80 ± 10% humidity, and offered 10% sucrose ad libitum. Mosquito survival was monitored daily for fourteen days and any dead mosquitoes were removed by aspiration and recorded. Fourteen days after the blood meal any remaining mosquitoes were recorded as alive and then frozen. The LC50 and LC90 of mosquitoes were estimated using a normalized concentration–response analysis (IC50 and Hill), assuming a maximum of 100% mosquito mortality and an estimated baseline mosquito mortality (i.e., mosquito mortality at zero drug concentration). The median time to death were calculated for mosquitoes subjected to 1 ± 0.5 ng/mL and 5 ± 0.5 ng/mL of ivermectin, using both clinical data and in vitro data. The median time to death for each unique ivermectin concentration was also calculated and data evaluated with a normalized concentration–response analysis (TC50 and Hill), assuming a maximum time to mosquito mortality of 10 days and an estimated baseline rate of mosquito mortality (i.e., rate of mosquito mortality at zero drug concentration). A similar analysis was conducted when comparing median time to death for ivermectin and its metabolites, assuming a maximum time to mosquito mortality of 14 days and a fixed hill-slope of an average value of -2.0 to facilitate model fitting. All mosquito survival analyses were performed with GraphPad Prism v.9.0 (GraphPad Software Inc, San Diego, CA, USA).
Ivermectin and metabolite quantification by LC–MS/MS
Samples (100 µL) was aliquoted into a 1-mL 96-well extraction plate followed by the addition of cold extraction solution (450 µL of 90% acetonitrile in water containing 15 ng/mL internal standard ivermectin-d2) into each well using a multi-dispenser pipette. The mixture was mixed on a MixMate set to 1000 rpm for 10 min. The suspension was then centrifuged at 2100×g for 10 min at 4 °C to separate the precipitates. The clear supernatant (380 µL) was transferred onto a HybridSPE® Plus 96-well plate (P/N 575659-U, Supelco, Darmstadt, Germany) to remove both precipitated proteins and phospholipids. 10 mM ammonium formate in water containing 0.1% formic acid (100 µL) was used to extract the sample compounds into the receiving plate. The final extract was mixed at 1000 rpm for 5 min and centrifuged at 1100×g for 5 min at 4 °C before the LC–MS/MS analysis.
LC–MS/MS analysis was carried out using a Thermo Scientific (Germering, Germany) Dionex Ultimate 3000 UHPLC system, coupled with Sciex (Woodlands, Singapore) triple quadrupole 6500 + mass spectrometer. The Ultimate 3000 UHPLC was equipped with an Acquity HSS T3 (2.1 × 100 mm, 1.8 µm, Waters (Milford, MA)) with a 5 mm Vanguard pre-column (Waters) of the same internal diameter, particle size, and type. Flow rate was maintained at 0.5 mL/min. The mobile phase contained (A) 10 mM ammonium formate in ultrapure water and (B) 5:95 (v/v) 10 mM ammonium formate in water/acetonitrile, both A and B with 0.1% formic acid. Gradient elution started at 75% B, ramped to 90% B in 3 min, and held constant for 6 min. The gradient returned to 75% B in 0.1 min with 2 min re-equilibration until the next injection. A divert valve switched flow from waste to the MS/MS after 2.4 min until 7 min. The total run time from injection to injection was approximately 11 min. The autosampler tray was set at 4 °C, column temperature was 40 °C, and injection volume was 5 µL.
Electrospray ionization (ESI) operated in positive mode and multiple reaction monitoring (MRM) was used in MS/MS. Three transitions were monitored for each targeted analyte9. Dwell time was set at 0.15 s to yield greater than six points for each chromatographic ion peak. Table S3 lists the MS/MS conditions for each analyte in order of retention times (tR). Curtain gas setting was 30 psi, source gases 1 and 2 settings were 40 psi and 55 psi, respectively, medium collision gas setting with ion spray voltage at + 5000 V, and the source temperature gas setting was 225 °C. Sciex Analyst 1.7.2 software (Woodlands, Singapore) was used for both instrument control data processing.
The LLOQ was determined for each analyte combination based on accuracy and precision data after samples were taken through the entire workflow including sample extraction and LC–MS/MS analysis. LLOQs were typically 0.25 ng/mL for all targeted analytes.
All determinations for individual ivermectin, M1 and M3 metabolites were calibrated using multipoint, calibration standards in whole blood, which bracketed the quality control (QC) samples. QC samples of ivermectin, M1 and M3 at four concentration levels were processed (triplicates at each level) simultaneously with the mosquito-blood meal samples and measured in a single analytical series, in order to ensure the overall method performance and the quality of reported drug concentrations. Due to the limitation of M6 reference standard material amounts available at the time of analysis, the calibration curve and other internal quality control parameters of the ivermectin parent drug were used to assess concentration measurements of M6.
Analyte identification in sample extracts was accomplished via tR and the presence of monitoring trace MRMs.
