Chemiluminescent Nitrogen Detection (CLND) to Measure Kinetic Aqueous Solubility

Aimee Kestranek1, Andrew Chervenak1, Justin Longenberger1, Steven Placko1

1 Analiza, Inc, Cleveland, Ohio
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130145
Online Posting Date:  December, 2013
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Solubility is the dose‐limiting property for in vitro studies, and therefore is a critical physicochemical property to measure in drug discovery. Solubility data can be used to guide lead optimization, troubleshoot erratic bioassay results, and identify potential downstream liabilities such as insufficient solubility for bioassays or oral bioavailability. Typically, early in vitro studies are performed using library compounds prepared as dimethylsulfoxide (DMSO) stock solutions, resulting in in vitro test solutions containing DMSO at low concentration (<5% v/v). Since DMSO can affect the apparent solubility, it is desirable to obtain solubility data under conditions mimicking the in vitro study. Kinetic solubility (from DMSO stock solutions) is often preferred over thermodynamic solubility (from dry powder) in early drug discovery. The protocols in this article describe a general procedure for assessing kinetic aqueous solubility of early drug discovery compounds using a miniaturized shake flask method with chemiluminescent nitrogen detection (CLND). Curr. Protoc. Chem. Biol. 5:269‐280 © 2013 by John Wiley & Sons, Inc.

Keywords: solubility; drug discovery; chemiluminescent nitrogen detection; physicochemical properties; shake flask; nephelometry

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Dimethylsulfoxide (DMSO) (ideally the same used to prepare the test article stock solutions)
  • Test articles as 10 mM DMSO stock solutions
  • Control compounds as 10 mM DMSO stock solutions: imipramine (Sigma‐Aldrich, cat no. I7379‐5G) and benzthiazide (Sigma‐Aldrich, cat. no. B7149‐1G)
  • Assay medium (see recipe)
  • Calibration standards (see recipe)
  • Sources of O 2 and Ar
  • Methanol
  • Mobile phase: 75:25 (v/v) methanol:water
  • 96‐well microtiter plate (Corning, cat. no. 3365)
  • 96‐well filter plate, 0.45 µM, polycarbonate (Millipore, cat. no. MSSLBPC10)
  • EZ‐Peel plate seals (Thermo Scientific, cat. no. AB‐0745)
  • Rotary plate shaker
  • 96‐well deep‐well collection plates (Arctic White, cat. no. 238984;
  • Vacuum manifold (Millipore, cat. no MSVMHT500)
  • EZ‐Pierce plate seals (Thermo Scientific, cat. no. AB‐0757)
  • CLND Workstation (PAC; consisting of a microplate autosampler, isocratic HPLC pump, CLND (cat. no. 8060), and data acquisition software
  • Graphing and curve‐fitting software: e.g., Table Curve 2D (Systat Software, Inc.,
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Literature Cited

Literature Cited
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