Use of Label-Free Pro-Drug, Stable-Labeled Drug and LC-MS/MS to Decouple the Pharmacokinetics of Drug and Pro-Drugs in the Same Animal, presented at the 2012 AAPS Annual Meeting and Exposition, Oct. 14-18, 2012 in Chicago, IL.

Purpose: In preliminary mouse PK studies with Riluzole pro-drugs, the IV clearance estimate for both the drug and the pro-drug were equivalent. Traditional mouse PK study designs using a mouse per
time point would not have the resolution to determine the effect of the pro-drug on the clearance of
Riluzole where the differences are within the range of animal variability. An experimental design was
required that could decouple the clearance of the drug from that of the pro-drug in a single animal.
Method: In order to decouple the clearance of Riluzole from the clearance of the pro-drug, CD-1 mice were co-dosed intravenously and orally with label-free pro-drug and stable isotope-labeled (SIL) drug.
At the specified time intervals over a period of 24 hours, whole blood samples (10 μL) were collected
via the tail vein and mixed with a small volume (1 μL) of EDTA as the anticoagulant. The pro-drug,
released drug and SIL-drug were isolated from mouse whole blood by protein removal and quantified
by LC-MS/MS in the positive ionization mode. An ABSciex API 5500 QTrap Mass Spectrometer
interfaced via the ABSciex Turbo V IonSpray source (ESI) to an Eksigent ExpressHT LC system was
Results: In this investigation, the assay using 10 μL of mouse blood was sensitive to 5-10 ng/mL,
and accurate to better than 80% of nominal. Replicate quality control samples showed very good
reproducibility, with precision better than 20% CV, for pro-drug, drug, and SIL-drug. The use of the
SIL-drug allowed decoupling of drug clearance from pro-drug cleavage, and the use of serial microsampling
provided the experimental resolution necessary to show the pro-drug decreased Riluzole
Conclusions: The combined approach presented here produces accurate and precise analytical data,
eliminates intra-subject variability, reduces inter-subject variability in mouse studies, saves analytical
and animal resources, and de-couples the PK of drug from that of pro-drug in a single experiment.


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