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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1:

  Materials
  • 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; http://www.arcticwhiteusa.com/)
  • Vacuum manifold (Millipore, cat. no MSVMHT500)
  • EZ‐Pierce plate seals (Thermo Scientific, cat. no. AB‐0757)
  • CLND Workstation (PAC; http://www.paclp.com/) 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., http://www.systat.com/)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Avdeef, A. 2001. Pharmacokinetic optimization in drug research: Biological, physicochemical, and computational strategies, LogP2000 Symposium, 2nd, Lausanne, Switzerland, March 5‐9, 2000, pp. 305‐325. John Wiley & Sons, New York.
  Bard, B., Martel, S., and Carrupt, P. 2008. High throughput UV method for the estimation of thermodynamic solubility and the determination of the solubility in biorelevant media. Eur. J. Pharm. Sci. 33:230‐240.
  Bevan, C.D. and Lloyd, R.S. 2000. A high‐throughput screening method for the determination of aqueous drug solubility using laser nephelometry in microtiter plates. Anal. Chem. 72:1781‐1787.
  Bhattachar, S., Wesley, J., and Seadeek, C. 2005. Evaluation of the chemiluminescent nitrogen detector for solubility determinations to support drug discovery. J. Pharm. Biomed. Anal. 41:152‐157.
  Borny, J.A. and Homan, M.E. 1999. Efficient characterization of combinatorial libraries using mass spectrometry and chemiluminescent nitrogen detection. LC GC Europe 5:S14.
  Chen, T.M., Shen, H., and Zhou, C. 2002. Evaluation of a method for high throughput solubility determination using a multi‐wavelength UV plate reader. Comb. Chem. High Thr. Screen. 5:575‐581.
  Di, L. and Kerns, E. 2006. Biological assay challenges from compound solubility: Strategies for bioassay optimization. Drug Discov. Today 11:446‐451.
  Kerns, E. and Di, L. 2008. Drug‐Like Properties: Concepts, Structure Design and Methods: from ADME to Toxicity Optimization, Elsevier Academic Press, Oxford.
  Lewis, K., Phelps, D., and Sefler, A. 2000. Automated high‐throughput. quantification of combinatorial arrays. Am. Pharm. Rev. 3:63‐68.
  Lipinski, C.A. 2004. Solubility in water and DMSO: Issues and potential solutions. In Pharmaceutical Profiling in Drug Discovery for Lead Selection, (R.T. Bochardt, E.H. Kerns, C.A. Lipinski, D.R. Thakker, and B. Wang, eds.), pp. 93‐126. AAPS Press, Arlington, Va.
  Lipinski, C.A., Lombardo, F., Dominy, B.W., and Feeney, P.J. 1997. Experimental and computational approaches to estimate solubility and permeability in drug discovery settings. Adv. Drug Deliv. Rev. 23:3‐25.
  Popa‐Burke, I.G., Issakova, O., Arroway, J.D., Bernasconi, P., Chen, M., Coudurier, L., Galin, S., Jadhav, A.P., Janzen, W.P., Lagasca, D., Liu, D., Lewis, R.S., Mohney, R.P., Sepetov, N., Sparkman, D.A., and Hodge, C.N. 2004. Streamlined system for purifying and quantifying a diverse library of compounds and the effect of compound concentration measurements on the accurate interpretation of biological assay results. Anal. Chem. 76:7278‐7287.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library