In Vivo Rapid Assessment of Compound Exposure (RACE) for Profiling the Pharmacokinetics of Novel Chemical Probes

Danielle McAnally1, Michael Vicchiarelli2, Khandaker Siddiquee1, Layton H. Smith1

1 Cardiovascular Pathobiology Program, Sanford‐Burnham Medical Research Institute, Orlando, Florida, 2 Conrad Prebys Center for Chemical Genomics, Sanford‐Burnham Medical Research Institute, Orlando, Florida
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch120072
Online Posting Date:  December, 2012
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The Rapid Assessment of Compound Exposure (RACE) assay is an easy and efficient method for estimating the pharmacokinetic parameter of exposure (AUC: area under the curve) of novel chemical probe compounds in mice. RACE is a truncated and compressed version of a traditional comprehensive in vivo pharmacokinetics study. The method uses a single standard formulation, dose, route of administration, and a small cohort of mice (n = 4). Standardized protocols and an abbreviated sample collection scheme reduce the labor needed to perform both the in‐life and bioanalytical phases of the study. The procedure reduces the complexity of data analysis by eliminating all but one calculated pharmacokinetic parameter; estimated exposure (eAUC20‐120), a parameter that is sufficient to rank order compounds based on exposure, but is also easily determined by most software using the simple trapezoidal rule. The RACE assay protocol is readily applicable to early/exploratory studies of most compounds, and is intended to be employed by laboratories with limited expertise in pharmacology and pharmacokinetics. Curr. Protoc. Chem. Biol. 4:299‐309 © 2012 by John Wiley & Sons, Inc.

Keywords: pharmacokinetics; exposure; rapid; in vivo; high‐throughput

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Rapid Assessment of Compound Exposure (RACE) Approach to Dosing and Blood Collection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Rapid Assessment of Compound Exposure (RACE) Approach to Dosing and Blood Collection

  • Mice [e.g., male, C57BL6 or BALBc (or other strain relevant to the research question), ∼25 g, 8 to 10 weeks old]
  • Vehicle: 10:10:80 (v/v/v) DMSO:TWEEN80:H 2O solution (see recipe)
  • Compound (e.g., small molecule or biologic)
  • Ice
  • Control mouse plasma (Bioreclamation, cat. no. MSEPLEDTA3‐C57); divide into aliquots and store up to 1 year at −80°C
  • Acetonitrile (Sigma, cat. no. 34967‐4L, LC‐MS Chromasolv)
  • Indomethacin, 99.9% (TLC) (Sigma, cat. no. I7378‐10G)
  • Balance (Mettler Toledo, cat. no. XP205)
  • Ear punch or tag
  • Sterile 1‐ml syringe with 23‐G sterile needle (BD, cat. no. 305145)
  • Sterile Goldenrod 5.0‐mm lancets (Medipoint, cat. no. Goldenrod5)
  • Sterile EDTA blood collection tubes (Sarstedt, cat. no. 16.444.100)
  • Gauze
  • Centrifuge (e.g., Eppendorf 5617R)
  • API 4000 LC/MS/MS (AB Sciex)
  • 1.7‐ml microcentrifuge tubes
  • Vortex mixer
  • 96‐well, 2‐ml plates
  • Acquity UPLC autosampler (Waters Corporation)
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Literature Cited

Literature Cited
   Berman, J., Halm, K., Adkison, K., and Shaffer, J. 1997. Simultaneous pharmacokinetic screening of a mixture of compounds in the dog using API LC/MS/MS analysis for increased throughput. J. Med. Chem. 40:827‐829.
   Golde, W.T., Gollobin, P., and Rodriguez, L.L. 2005. A rapid, simple, and humane method for submandibular bleeding of mice using a lancet. Lab. Anim. 34:39‐43.
   Korfmacher, W.A., Cox, K.A., Ng, K.J., Veals, J., Hsieh, Y., Wainhaus, S., Broske, L., Prelusky, D., Nomeir, A., and White, R.E. 2001. Cassette‐accelerated rapid rat screen: a systematic procedure for the dosing and liquid chromatography/atmospheric pressure ionization tandem mass spectrometric analysis of new chemical entities as part of new drug discovery. Rapid Comm. Mass Spectr. 15:335‐340.
   Lee, Y.C., Zocharski, P.D., and Samas, B. 2003. An intravenous formulation decision tree for discovery compound formulation development. Int. J. Pharm. 253:111‐119.
   Liu, B., Chang, J., Gordon, W.P., Isbell, J., Zhou, Y., and Tuntland, T. 2008. Snapshot PK: A rapid rodent in vivo preclinical screening approach. Drug Discov. Today 13:360‐367.
   White, R.E. and Manitpisitkul, P. 2001. Pharmacokinetic theory of cassette dosing in drug discovery screening. Drug Metab. Dispos. 29:957‐966.
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