Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

High-throughput microplate assay for the determination of drug partition coefficients

Nature Protocols volume 5, pages 1823–1830 (2010)Cite this article

Subjects

Abstract

Partition coefficients (Kp) of drugs between the phospholipid bilayer and the aqueous phase provide useful information in quantitative structure-activity relationship studies. Hexadecylphosphocholine (HePC) micelles, composed of a zwitterionic hydrophilic surface and a hydrophobic core, mimic the biomembranes and have several advantages over other lipid structures to assess Kp values. Their preparation is easy, fast and avoids the use of toxic organic solvents, and the output has fewer spectroscopic interferences. Here, we describe a high-throughput microplate protocol for assessing the Kp of drugs using HePC micelles as membrane models and derivative spectrophotometry as the detection technique. Moreover, the time-consuming data treatment to assess Kp values is easily performed by a dedicated Excel routine developed here and described in detail. The Kp values of nonsteroidal anti-inflammatory drugs (acemetacin, clonixin, diclofenac and indomethacin) were determined to show the simplicity of the method and to validate this protocol, which provides Kp values (n = 3) of two drugs in ∼2 h.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Standard curves used to determine a suitable drug concentration for the assay.
Figure 2: Representation of the 96-well microplate used in this protocol.
Figure 3: Screenshot of the 'Exp. Results' sheet in Kp Calculator.
Figure 4: Schematic representation of HePC micelle–buffer partition coefficient of indomethacin calculated by Kp Calculator.

Similar content being viewed by others

References

  1. Di, L. & Kerns, E.H. Profiling drug-like properties in discovery research. Curr. Opin. Chem. Biol. 7, 402–408 (2003).

    Article  CAS  Google Scholar 

  2. Seydel, J.K. & Wiese, M. Drug-Membrane Interactions: Analysis, Drug Distribution, Modeling (Wiley-VCH Verlag, Weinheim, 2002).

  3. Avdeef, A. Physicochemical profiling (solubility, permeability and charge state). Curr. Top. Med. Chem. 1, 277–351 (2001).

    Article  CAS  Google Scholar 

  4. Hansch, C. & Dunn, W.J. Linear relationship between lipophilic character and biological activity of drugs. J. Pharm. Sci. 61, 1–19 (1972).

    Article  CAS  Google Scholar 

  5. Betageri, G.V. & Rogers, J.A. The liposome as a distribution model in QSAR studies. Int. J. Pharm. 46, 95–102 (1988).

    Article  CAS  Google Scholar 

  6. Hansch, C., Leo, A., Mekapati, S.B. & Kurup, A. QSAR and ADME. Bioorg. Med. Chem. 12, 3391–3400 (2004).

    Article  CAS  Google Scholar 

  7. Schultz, T.W., Cronin, M.T.D., Walker, J.D. & Aptula, A.O. Quantitative structure-activity relationships (QSARs) in toxicology: a historical perspective. Theochem. J. Mol. Struct. 622, 1–22 (2003).

    Article  CAS  Google Scholar 

  8. Klopman, G. & Zhu, H. Recent methodologies for the estimation of N-octanol/water partition coefficients and their use in the prediction of membrane transport properties of drugs. Mini Rev. Med. Chem. 5, 127–133 (2005).

    Article  CAS  Google Scholar 

  9. Oprea, T.I., Davis, A.M., Teague, S.J. & Leeson, P.D. Is there a difference between leads and drugs? A historical perspective. J. Chem. Inf. Comput. Sci. 41, 1308–1315 (2001).

    Article  CAS  Google Scholar 

  10. Ong, S.W., Liu, H.L., Qiu, X.X., Bhat, G. & Pidgeon, C. Membrane partition coefficients chromatographically measured using immobilized artificial membrane surfaces. Anal. Chem. 67, 755–762 (1995).

    Article  CAS  Google Scholar 

  11. Escher, B.I. & Schwarzenbach, R.P. Partitioning of substituted phenols in liposome-water, biomembrane-water, and octanol-water systems. Environ. Sci. Technol. 30, 260–270 (1996).

    Article  CAS  Google Scholar 

  12. Wang, M.M., Sugar, I.P. & Chong, P.L.G. Role of the sterol superlattice in the partitioning of the antifungal drug nystatin into lipid membranes. Biochemistry 37, 11797–11805 (1998).

    Article  CAS  Google Scholar 

  13. de Castro, B., Gameiro, P., Lima, J.L.F.C., Matos, C. & Reis, S. A fast and reliable spectroscopic method for the determination of membrane-water partition coefficients of organic compounds. Lipids 36, 89–96 (2001).

    Article  CAS  Google Scholar 

  14. Takegami, S., Kitamura, K., Funakoshi, T. & Kitade, T. Partitioning of anti-inflammatory steroid drugs into phosphatidylcholine and phosphatidylcholine-cholesterol small unilamellar vesicles as studied by second-derivative spectrophotometry. Chem. Pharm. Bull. 56, 663–667 (2008).

    Article  CAS  Google Scholar 

  15. Omran, A.A. et al. Effect of phosphatidylserine content on the partition coefficients of diazepam and flurazepam between phosphatidyleholine-phosphatidylserine bilayer of small unilamellar vesicles and water studied by second derivative spectrophotometry. Chem. Pharm. Bull. 50, 312–315 (2002).

    Article  CAS  Google Scholar 

  16. Chakraborty, H. & Sarkar, M. Interaction of piroxicam and meloxicam with DMPG/DMPC mixed vesicles: anomalous partitioning behavior. Biophys. Chem. 125, 306–313 (2007).

    Article  CAS  Google Scholar 

  17. Ferreira, H., Lucio, M., Lima, J.L.F.C., Matos, C. & Reis, S. Effects of diclofenac on EPC liposome membrane properties. Anal. Bioanal. Chem. 382, 1256–1264 (2005).

    Article  CAS  Google Scholar 

  18. Ferreira, H., Lucio, M., Lima, J.L.F.C., Matos, C. & Reis, S. Interaction of clonixin with EPC liposomes used as membrane models. J. Pharm. Sci. 94, 1277–1287 (2005).

    Article  CAS  Google Scholar 

  19. Lucio, M. et al. Effect of anti-inflammatory drugs in phosphatidylcholine membranes: a fluorescence and calorimetric study. Chem. Phys. Lett. 471, 300–309 (2009).

    Article  CAS  Google Scholar 

  20. Lucio, M. et al. Influence of some anti-inflammatory drugs in membrane fluidity studied by fluorescence anisotropy measurements. Phys. Chem. Chem. Phys. 6, 1493–1498 (2004).

    Article  CAS  Google Scholar 

  21. Tanaka, A., Nakamura, K., Nakanishi, I. & Fujiwara, H. A novel and useful descriptor for hydrophobicity, partition coefficient micellar-water, and its application to a QSAR study of antiplatelet agents. J. Med. Chem. 37, 4563–4566 (1994).

    Article  CAS  Google Scholar 

  22. de Castro, B., Gameiro, P., Lima, J.L.F.C., Matos, C. & Reis, S. Interaction of drugs with hexadecylphosphocholine micelles. Derivative spectroscopy, acid-base and solubility studies. Mat. Sci. Eng. C 18, 71–78 (2001).

    Article  Google Scholar 

  23. Cudina, O., Brboric, J., Jankovic, I., Karljikovic-Rajic, K. & Vladimirova, S. Study of valsartan interaction with micelles as a model system for biomembranes. Colloids Surf. B Biointerfaces 65, 80–84 (2008).

    Article  CAS  Google Scholar 

  24. Marcelino, J., Lima, J.L.F.C., Reis, S. & Matos, C. Assessing the effects of surfactants on the physical properties of liposome membranes. Chem. Phys. Lipids 146, 94–103 (2007).

    Article  CAS  Google Scholar 

  25. Santos, N.C., Prieto, M. & Castanho, M.A.R.B. Quantifying molecular partition into model systems of biomembranes: an emphasis on optical spectroscopic methods. Biochim. Biophys. Acta 1612, 123–135 (2003).

    Article  CAS  Google Scholar 

  26. Kitamura, K. et al. Second-derivative spectrophotometric determination of partition coefficients of chlorpromazine and promazine between lecithin bilayer vesicles and water. Anal. Chim. Acta 304, 101–106 (1995).

    Article  CAS  Google Scholar 

  27. Ferreira, H. et al. Partition and location of nimesulide in EPC liposomes: a spectrophotometric and fluorescence study. Anal. Bioanal. Chem. 377, 293–298 (2003).

    Article  CAS  Google Scholar 

  28. Omran, A.A., Kitamura, K., Takegami, S., El-Sayed, A.A.Y. & Abdel-Mottaleb, A. Determination of partition coefficients of diazepam and flurazepam between phosphatidylcholine bilayer vesicles and water by second derivative spectrophotometric method. J. Pharm. Biomed. Anal. 25, 319–324 (2001).

    Article  CAS  Google Scholar 

  29. Ojeda, C.B. & Rojas, E.S. Recent developments in derivative ultraviolet/visible absorption spectrophotometry. Anal. Chim. Acta 518, 1–24 (2004).

    Article  Google Scholar 

  30. Savitzky, A. & Golay, M.J.E. Smoothing and differentiation of data by simplified least squares procedures. Anal. Chem. 36, 1627–1634 (1964).

    Article  CAS  Google Scholar 

  31. Pola, A., Michalak, K., Burliga, A., Motohashi, N. & Kawase, M. Determination of lipid bilayer/water partition coefficient of new phenothiazines using the second derivative of absorption spectra method. Eur. J. Pharm. Sci. 21, 421–427 (2004).

    Article  CAS  Google Scholar 

  32. Videira, R.A., Antunes-Madeira, M.D., Custodio, J.B.A. & Madeira, V.M.C. Partition of DDE in synthetic and native membranes determined by ultraviolet derivative spectroscopy. Biochim. Biophys. Acta Biomembr. 1238, 22–28 (1995).

    Article  Google Scholar 

Download references

Acknowledgements

L.M.M. thanks FSE (Fundo Social Europeu) and MCTES (Ministério da Ciência, Tecnologia e Ensino Superior) for the financial support through the POPH-QREN program. C.N. thanks FCT (Fundação para a Ciência e Tecnologia) for the PhD grant (SFRH/BD/38445/2007). This work was financially supported by FCT through project PTDC/SAU-FCF/67718/2006.

Author information

Authors and Affiliations

  1. Departamento de Química, REQUIMTE, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, Porto, Portugal

    Luís M Magalhães, Cláudia Nunes, Marlene Lúcio, Marcela A Segundo, Salette Reis & José L F C Lima

Authors
  1. Luís M Magalhães
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cláudia Nunes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marlene Lúcio
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marcela A Segundo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Salette Reis
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. José L F C Lima
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All the authors contributed extensively to the work presented in this article.

Corresponding author

Correspondence to Salette Reis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Method 1 (Excel 2007)

Kp Calculator, for the determination of drug partition coefficients. Kp Calculator as an xltm file. Use for Office 2007 and superior. (ZIP 2117 kb)

Supplementary Method 2 (Excel 2003)

Kp Calculator, for the determination of drug partition coefficients. Kp Calculator as an xls file. Use for Office 2003. (XLS 7315 kb)

Supplementary Method 3

Installation procedures of Kp Calculator, word file that explains the installation steps. (DOC 35 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Magalhães, L., Nunes, C., Lúcio, M. et al. High-throughput microplate assay for the determination of drug partition coefficients. Nat Protoc 5, 1823–1830 (2010). https://doi.org/10.1038/nprot.2010.137

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.137

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research