TFV-DP adherence interpretations from TDF using an updated 50% methanol and 50% water extraction method for DBS

Abstract

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TFV-DP adherence interpretations from TDF using an updated 50% methanol and 50% water extraction method for DBS

Corwin Coppinger¹, Mary Morrow¹, Samantha MaWhinney¹, Martin Williams¹, Lane Bushman¹, Kristina Brooks¹, Kenneth Mugwanya^1,2 and Peter Anderson¹

¹University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences; ²Department of Global Health, University of Washington

Background: Intraerythrocytic tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) in dried blood spots (DBS) have been used in many clinical trials to better understand cumulative and recent adherence, respectively. The original validated extraction utilized 70% methanol and 30% water (70:30), which required controlled extraction conditions for reproducible results. We recently validated a 50% methanol and 50% water extraction (50:50), which yields better and more reproducible drug recoveries with fewer limitations on the control of extraction conditions. The aim of this study was to compare the extraction performances (70:30 vs. 50:50) and to adjust the original TFV-DP interpretations, which was based on 70:30, using the 50:50 extraction process.

Methods: DBS from the Benchmark study were used for this analysis. The benchmark study included 53 African cisgender women without HIV randomized to two, four or seven doses per week, directly observed, for 8 weeks. An additional 17 pregnant women received 7 doses/week for 8 weeks. DBS samples were collected weekly; each sample included five 50uL spots. Three hundred ninety-six samples were available for this analysis. Both extraction methods were run in parallel to assess the relative efficiency of extracting TFV-DP and FTC-TP: Two 3-mm punches were removed from the same 50 μL spot from each card, and one punch was extracted with 70:30 and the other with 50:50. TFV-DP and FTC-TP concentrations were quantified using validated LC-MS/MS. A linear regression on the logarithmic scale was used to compare the results from the two extraction methods. The fold difference between extraction methods was applied to the original 70:30 TFV-DP adherence interpretations, which were <350 (<2 dose/week), 350–699 (2–3 doses/week), 700–1249 (4–6 doses/week) and ≥1250 fmol/punch (7 doses/week). These were generated from the DOT-DBS study conducted in the United States.

Results: Data from the 70:30 extraction were within 10% of the original 70:30 TFV-DP adherence table estimates based on DOT-DBS, validating these interpretations for African cisgender women. The 50:50 extraction resulted in 1.27 (95% CI 1.25, 1.28) higher TFV-DP concentrations compared with the 70:30 extraction. The conversion factor of 1.27 was applied to the previous 70:30 TFV-DP benchmarks to produce the following interpretations for 50:50 extraction: <450 (<2 dose/week), 450–899 (2–3 doses/week), 900–1599 (4–6 doses/week) and ≥1600 fmol/punch (7 doses/week).

Conclusions: This study used samples from a directly observed dosing study in cisgender African women to demonstrate that 70:30 extraction matched previous TFV-DP adherence interpretations. The new 50:50 extraction resulted in 1.27-fold higher recoveries enabling the establishment of a new interpretation table for 50:50 extraction. Future studies can use either table for adherence assessment based on the extraction used.