The AAPS Journal

, Volume 12, Issue 3, pp 318–329 | Cite as

Identification of Two Novel, Potent, Low-Liability Antinociceptive Compounds from the Direct In Vivo Screening of a Large Mixture-Based Combinatorial Library

  • Kate J. Reilley
  • Marc Giulianotti
  • Colette T. Dooley
  • Adel Nefzi
  • Jay P. McLaughlin
  • Richard A. Houghten
Research Article Theme: Fishing for the Hidden Proteome in Health and Disease: Focus on Drug Abuse


Synthetic combinatorial methods now make it practical to readily produce hundreds of thousands of individual compounds, but it is clearly impractical to screen each separately in vivo. We theorized that the direct in vivo testing of mixture-based combinatorial libraries during the discovery phase would enable the identification of novel individual compounds with desirable antinociceptive profiles while simultaneously eliminating many compounds with poor absorption, distribution, metabolism, or pharmacokinetic properties. The TPI 1346 small-molecule combinatorial library is grouped in 120 mixtures derived from 26 functionalities at the first three positions and 42 functionalities at the fourth position of a pyrrolidine bis-cyclic guanidine core scaffold, totaling 738,192 compounds. These 120 mixtures were screened in vivo using the mouse 55°C warm water tail-withdrawal assay to identify mixtures producing antinociception. From these data, two fully defined individual compounds (TPI 1818-101 and TPI 1818-109) were synthesized. These were examined for antinociceptive, respiratory, locomotor, and conditioned place preference effects. The tail-withdrawal assay consistently demonstrated distinctly active mixtures with analgesic activity that was blocked by pretreatment with the non-selective opioid antagonist, naloxone. Based on these results, synthesis and testing of TPI 1818-101 and 1818-109 demonstrated a dose-dependent antinociceptive effect three to five times greater than morphine that was antagonized by mu- or mu- and kappa-opioid receptor selective antagonists, respectively. Neither 1818-101 nor 1818-109 produced significant respiratory depression, hyperlocomotion, or conditioned place preference. Large, highly diverse mixture-based libraries can be screened directly in vivo to identify individual compounds, potentially accelerating the development of promising therapeutics.

Key words

analgesia in vivo mixture-based libraries opioid testing 



Blood–brain barrier


Central nervous system


Conditioned place preference




Delta-opioid receptor








Kappa-opioid receptor


Mu-opioid receptor











This work was supported by NIDA grant R21 DA019620 (to RAH) and by the State of Florida, Executive Office of the Governor’s Office of Tourism, Trade, and Economic Development.


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Copyright information

© American Association of Pharmaceutical Scientists 2010

Authors and Affiliations

  • Kate J. Reilley
    • 1
  • Marc Giulianotti
    • 1
  • Colette T. Dooley
    • 1
  • Adel Nefzi
    • 1
  • Jay P. McLaughlin
    • 1
  • Richard A. Houghten
    • 1
  1. 1.Torrey Pines Institute for Molecular StudiesPort St. LucieUSA

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