Quantification of Tafenoquine and 5,6-Orthoquinone Tafenoquine by UHPLC-MS/MS in Blood, Plasma, and Urine, and Application to a Pharmacokinetic Study

Abstract

Analytical methods for the quantification of the new 8-aminoquinoline antimalarial tafenoquine (TQ) in human blood, plasma and urine, and the 5,6-orthoquinone tafenoquine metabolite (5,6-OQTQ) in human plasma and urine have been validated. The procedure involved acetonitrile extraction of samples followed by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Chromatography was performed using a Waters Atlantis T3 column with a gradient of 0.1% formic acid and acetonitrile at a flow rate of 0.5 mL per minute for blood and plasma. Urine analysis was the same but with methanol containing 0.1% formic acid replacing acetonitrile mobile phase. The calibration range for TQ and 5,6-OQTQ in plasma was 1 to 1200 ng/mL, and in urine was 10 to 1000 ng/mL. Blood calibration range for TQ was 1 to 1200 ng/mL. Blood could not be validated for 5,6-OQTQ due to significant signal suppression. The inter-assay precision (coefficient of variation %) was 9.9% for TQ at 1 ng/mL in blood (n = 14) and 8.2% for TQ and 7.1% for 5,6-OQTQ at 1 ng/mL in plasma (n = 14). For urine, the inter-assay precision was 8.2% for TQ and 6.4% for 5,6-OQTQ at 10 ng/mL (n = 14). TQ and 5,6-OQTQ are stable in blood, plasma and urine for at least three months at both −80 °C and −20 °C. Once validated, the analytical methods were applied to samples collected from healthy volunteers who were experimentally infected with Plasmodium falciparum to evaluate the blood stage antimalarial activity of TQ and to determine the therapeutic dose estimates for TQ, the full details of which will be published elsewhere. In this study, the measurement of TQ and 5,6-OQTQ concentrations in samples from one of the four cohorts of participants is reported. Interestingly, TQ urine concentrations were proportional to parasite recrudescence times post dosing To our knowledge, this is the first description of a fully validated method for the measurement of TQ and 5,6-OQTQ quantification in urine.

Liver-targeted polymeric prodrugs delivered subcutaneously improve tafenoquine therapeutic window for malaria radical cure

“…S11). This metabolite pattern thus follows prior results for the oral TQ PK and metabolism in mouse models with low OQTQ concentration in plasma (55,56). The urine profile was also characterized, and a high level of OQTQ metabolite was found after pSVCTQ SC administration, consistent with a urine signature for this metabolite reported previously in human trials involving TQ (55).…”

“…Additional linker and polymer designs that slow the TQ release in hepatocytes could further reduce hemotoxicity by lowering the amount of drug recirculating into the blood. We have also characterized two key 8-AQ metabolites that have been extensively characterized with PQ in the past and to a lesser extent with TQ (38,(54)(55)(56)(57)(58)(59)85). The CTQ and OQTQ were not found at significant levels in the plasma, while they were both found in the liver.…”

“…Metabolites have been partially characterized previously, although the correlates of specific metabolite signatures for efficacy versus hemotoxicity are unfortunately not yet clearly available. Two key metabolites are carboxy-TQ (CTQ) and 5,6 orthoquinone-TQ (OQTQ), which have been previously characterized as markers of TQ metabolism in liver and blood (38,(54)(55)(56)(57)(58)(59). Both metabolites were found in liver at greater concentrations compared to plasma, with CTQ absence and low OQTQ in the plasma (fig.…”

Characterizing the blood stage antimalarial activity of tafenoquine in healthy volunteers experimentally infected withPlasmodium falciparum

“…preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 960 [16] Single doses of 1 to 40 mg/kg tafenoquine, with an increment of 1 mg/kg, were simulated in 20 trials of 100 patients with each body weight. Simulations were performed using the pharmacokinetic/pharmacodynamic model developed from the data obtained in healthy volunteers with induced blood stage P. falciparum.…”

“…This would limit its use for the purpose of clearing asexual parasitaemia in MDA where widespread G6PD screening would prove costly and logistically difficult. 960 [16] Single doses of 1 to 40 mg/kg tafenoquine, with an increment of 1 mg/kg, were simulated in 20 trials of 100 patients with each body weight. Simulations were performed using the pharmacokinetic/pharmacodynamic model developed from the data obtained in healthy volunteers with induced blood stage P. falciparum.…”