O-12: BUILDING LOCAL CAPACITY FOR HYDROXYUREA PHARMACOKINETICS AND PRECISION DOSING IN LOW-RESOURCE SETTINGS

Abstract

Purpose: Hydroxyurea is a highly effective disease-modifying treatment for sickle cell anemia (SCA) and designated an Essential Medicine by the WHO. Increasing access to hydroxyurea for SCA in low-resource settings is advocated, but the ideal approach to dosing and monitoring is debated. Escalation to maximum tolerated dose (MTD) achieves superior benefits without additional toxicities but requires incremental dose adjustments with serial monitoring. Pharmacokinetic (PK)-guided dosing uses a test dose and sparse blood sampling to model hydroxyurea metabolism and predict a personalized optimal dose, which should require fewer clinical visits, lab assessments, and dose adjustments. Currently PK-guided dosing requires expensive, complex analyses that are unavailable in low-resource settings. A simplified method for hydroxyurea PK analysis, coupled with a user-friendly dosing algorithm, could simplify dosing and monitoring. Materials and methods: Concentrated stock solutions of reagents for chemical detection of serum hydroxyurea using high performance liquid chromatography (HPLC) were stored at -80C. On the day of analysis, hydroxyurea was serially diluted in human serum, then spiked with N-methylurea as an internal standard. After deproteination with trichloroacetic acid, a Fearon reaction to detect the urea moiety was performed with diacetylmonoxime, ferric chloride, sulfuric acid, and phosphoric acid. Thiosemicarbazide was added to minimize photolability of the colored product, which was analyzed using two commercial HPLC machines: (1) a standard benchtop Agilent HPLC system with a 449nm detector and 4.6mm x 250mm, 5 micron Zorbax Eclipse XDB-C18 column; and (2) a portable PolyLC SmartLife HPLC system with a 415nm detector and 4.6mm x 50mm, 3.5 micron Zorbax column XDB-C18 column (Figure 1). The mobile phases were acetonitrile and water. After validation in the US, the portable HPLC and chemicals were transported to Tanzania for repeat analyses. Results: A calibration curve was generated using the concentration of hydroxyurea dilutions ranging from 0mM to 1000mM, plotted against the hydroxyurea:N-methylurea ratio, and both HPLC systems yielded calibration curves with R2>0.99 (Figure 1). A separate stock of hydroxyurea was diluted to create known concentrations for analysis using the calibration curve, to confirm accuracy and precision within 10-20% of the known value. Both HPLC systems measured hydroxyurea concentrations with <10% variance from the prepared concentration. Paired analysis of samples on both machines yielded results with <15% variance. Serial measurements of a standard 100 mM concentration using the PolyLC system were precise with a 2.5% coefficient of variance. After transport to Tanzania, setup, and training, the SmartLife HPLC system was able to produce similar excellent hydroxyurea concentration calibration curves with R2>0.99. Conclusion: Improving access to hydroxyurea as an essential treatment for all children living with SCA requires an approach that eases financial and logistical barriers while optimizing safety and benefit, especially in low-resource settings. We modified a portable HPLC instrument to detect hydroxyurea, validated its precision and accuracy, and confirmed capacity building and knowledge transfer to a low-resource setting. Implementation of HPLC for measurement of serum hydroxyurea concentrations is now feasible in low resource settings using available laboratory infrastructure. PK-dosing of hydroxyurea may soon be possible, with initial treatment using a personalized optimal dose.Agilent (unmodified) and SmartLife (modified) HPLC instruments for measuring serum hydroxyurea concentrationsCalibration curve of 0-1000 µM hydroxyurea measured with SmartLife PolyLC high performance liquid chromatography system demonstrating excellent R2 value in both the US (A) and Tanzania (B). The authors do not declare any conflict of interest