Schultze-Mosgau et al.: Comprehensive characterization of the PK of VPR (Clinical Pharmacokinetics)

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Journal: Clinical Pharmacokinetics

Characterization of the Pharmacokinetics of Vilaprisan: Bioavailability, Excretion, Biotransformation, and Drug-Drug Interaction Potential

Marcus-Hillert Schultze-Mosgau 1, Joachim Höchel 1, Olaf Prien 1, Torsten Zimmermann 1, Ashley Brooks2, Jim Bush 2, Antje Rottmann1

1 Bayer AG, Berlin, Germany
2 Covance Clinical Research Unit Ltd, Leeds, UK

Corresponding author:

Dr. M.-H. Schultze-Mosgau
E-mail:

Bioanalytical methods

Quantitation of vilaprisan in plasma (LC-MS/MS)

VPR was determined in lithium heparin plasma after addition of an internal standardbyprotein precipitation followed by liquid chromatography and tandem mass spectrometricdetection (LC-MS/MS).

All samples were stored at nominal -20°C and nominal -80°C and analyzed within 89 daysafter sampling. This is within the known stabilityof434days when vilaprisan is spiked inhuman plasmastored at nominal -20°Cand nominal-80°C.

Quantitation of itraconazole(ITZ) in plasma

ITZ was determined in human EDTA K3plasma samples. In brief, to a sample aliquot of 0.05mL, the internal standarditraconazole-d3was added, followed by an automated liquid-liquidextraction.Separationwasachievedbymeans of a liquid chromatographic system. Forthe mass spectrometric detection a triplequadrupole mass spectrometer in positiveTurboIonSprayTMionization mode was applied.

All samples were stored at -80 °C and were analyzed within the storage period of 42 days afterblood withdrawal. The stabilitydata indicated thatITZis stable in human EDTA K3plasmafor this time period. Thedemonstrated stabilityis 387 daysat -80 °C.

Liquid scintillation counting

For urine and feces samples, determination of [14C]-radioactivity amounts was based on the sample weights. Blood and plasma were analyzed by volume.

Feces were pooled per subject according to the appropriate 24 hour collection period. Pooled samples were homogenized in an appropriate volume of deionized water, avoiding excessive dilution. Triplicate aliquots of homogenate were submitted for combustion analysis. A sub-sample was retained for repeat analysis of total radioactivity which was stored at nominally -20°C. Additional 2 x ca. 50 g sub-samples were retained for metabolite profiling. Sub-samples for metabolite profiling were stored at nominally -70°C. A ca. 200 g aliquot (or as much remained if less the 200 g) of the remaining bulk feces homogenate were retained as a backup and was stored at nominally -20°C.

Portions of plasma (0.5 mL, in duplicate) and urine (1 mL, weighed aliquots in triplicate) were added directly to liquid scintillant prior to liquid scintillation counting. Plasma samples were analyzed using low potassium scintillation vials.

Portions of whole blood (ca. 400 μL) were added to flock and allowed to dry overnight in an oven at ca. 50°C prior to combustion analysis.

Feces and whole blood samples were combusted using a Packard Sample Oxidizer (Canberra Packard, Pangbourne, Berkshire, UK). The combusted products were absorbed in CarbosorbR (Perkin Elmer Life Sciences, Ltd,) and mixed with Permafluor E+ scintillation fluid (Perkin Elmer Life Sciences, Ltd). The efficiency of oxidation was determined by combustion of QC standards.

Radioactivity in excreta was measured for 5 minutes using a Packard TriCarb liquid scintillation counter (Canberra Packard) with the facilities for computing quench-corrected disintegrations per minute (dpm).

Prior to analysis, samples were allowed to stabilize with regard to light and temperature.

Efficiency correlation curves were prepared and routinely checked by the use of [14C]-toluene or [14C]-Ultima GoldTM quenched standards (supplied by PerkinElmer LAS (UK) Ltd). ProFlow G+ scintillant was supplied by Meridian Biotechnologies Ltd.

The LOQ of each batch of fecal samples or whole blood samples analyzed by combustion were taken as twice the mean background disintegration rate obtained when Combusto-ConesTM containing ashless floc were combusted.

Quantitation of [14C]vilaprisan in plasma

Plasma samples generated during the study were analyzed in two ways, direct accelerator massspectrometry (AMS) analysis for total carbon-14 concentration and a validated bioanalytical assayutilizing ultra-high performance liquid chromatography with detection using accelerator massspectrometry (UHPLC+AMS) for the quantification of [14C]vilaprisan. The parameters of theUHPLC+AMS assay are summarized in the following table.

Analyte: [14C]Vilaprisan

Internalstandard:Vilaprisan

Species:Human

Analyticalmatrix:Plasma(LithiumHeparinanti-coagulant)

Analyticalmethod:154-001.02

Calibrationrange:0.0672dpm/mL–6.01dpm/mL

0.502pg/mL*–44.9pg/mL*

Lowerlimitofquantification(LLOQ):0.0672dpm/mL

0.502pg/mL*

Qualitycontrol(QC)levels:Plasma:0.201,3.00,4.51dpm/mL

2.01dpm/mL(10xdilutionQC)

SampleVolume:Plasma:200μL

Calibrationmodel:Linearregression

Weightingfactor:1/x2

Analyticaltechnique/methodofdetection:UHPLC+AMS1

*based on the specific radioactivityof the [14C]vilaprisani.v. tracer infused during the study; certificate of analysisprovided by the sponsor (2.23 MBq/mg = 133,800,000 dpm/mg)

1The equipment and column utilized for this separation are appropriate for operation at pressures greater than 6000 PSI/ 400 bar, liquid chromatography at such high pressures can be referred to as ultra or ultra high-performance liquid chromatography(UPLC/UHPLC).