Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method Development and Pharmacokinetics of 10β,17β-Dihydroxyestra-1,4-dien-3-one (DHED) in Mice

Authors

• AQ Wang
• C Ryu
• M Hall
• E Padilha
• P Morris
• AF Postle
• CF Powels
• TD Gould
• X Xu

Abstract

10β,17β-dihydroxyestra-1,4-dien-3-one (DHED) is a prodrug of 17β-estradiol (E2) which releases 17β-estradiol within the central nervous system. DHED has been demonstrated to have neuroprotective and antidepressant effects in animal models, without peripheral estrogenic effects. We developed a sensitive and selective UPLC–MS/MS assay for the determination of DHED and E2 for ADME and pharmacokinetics (PK) studies.

Male CD-1 mice were dosed with DHED via IV (20 mg/kg) and PO (20 mg/kg) routes with plasma and brain tissue collected. Samples were prepared by protein precipitation. Supernatant was injected for UPLC-MS/MS analysis using negative ionization electrospray. A 2-min UPLC gradient method was developed using 0.1% ammonium in water and methanol as mobile phases.

The linear calibration range of DHED was 1-10,000 ng/mL (or ng/g tissue), and E2 was 0.5-1,000 ng/mL (or ng/g tissue). The accuracy of the QCs was between 85-115 % of the nominal value in both matrices. The optimized UPLC-MS/MS method provided good chromatographic retention for DHED and E2, reduced the sample preparation and analysis time, and increased sample throughput. Preliminary data indicated that DHED readily crossed the blood-brain barrier, with a brain to plasma AUC ratio of 0.4-0.7. DHED had short elimination half-lives ranging from 0.7-1.1 hr. Following 20 mg/kg IV administration of DHED, the plasma clearance was high (104 mL/min/kg) and the volume of distribution at steady-state was 0.9 L/kg. Based on the dose normalized AUC0-∞ values between the PO and the IV studies, the estimated bioavailability of DHED was 4% in mice.

Scientific Focus Area: Molecular Pharmacology

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