Skip to main content
SpringerLink
Log in

The Effect of Promiscuous Aggregation on in Vitro Drug Metabolism Assays

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

Many bioactive molecules show a type of solution phase behavior, termed promiscuous aggregation, whereby at micromolar concentrations, colloidal drug-rich aggregates are formed in aqueous solution. These aggregates are known to be a major cause of false positives and false negatives in select enzymatic high-throughput screening assays. The goal of this study was to investigate the impact of drug-rich aggregates on in vitro drug screening metabolism assays.

Methods

Cilnidipine was selected as an aggregate former and its impact on drug metabolism was evaluated against rCYP2D6, rCYP1A2, rCYP2C9 and human liver microsomes.

Results

The cilnidipine aggregates were shown to non-specifically inhibit multiple cytochrome P450 enzymes with an IC50 comparable with the IC50 of potent model inhibitors.

Conclusions

This newly demonstrated mode of “promiscuous inhibition” is of great importance as it can lead to false positives during drug metabolism evaluations and thus it needs to be considered in the future to better predict in vivo drug-drug interactions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

CAC:

Critical aggregation concentration

DDI:

Drug-drug interactions

HLM:

Human liver microsomes

HPLC:

High performance liquid chromatography

References

  1. Galetin A, Gertz M, Houston JB. Potential role of intestinal first-pass metabolism in the prediction of drug–drug interactions. Expert Opin Drug Metab Toxicol. 2008;4(7):909–22. https://doi.org/10.1517/17425255.4.7.909.

    Article  CAS  PubMed  Google Scholar 

  2. Bjornsson TD, Callaghan JT, Einolf HJ, Fischer V, Gan L, Grimm S, et al. The conduct of in vitro and in vivo drug-drug interaction studies: a pharmaceutical research and manufacturers of america (PhRMA) perspective. Drug Metab Dispos. 2003;31(7):815 LP–832.

    Article  Google Scholar 

  3. Bjornsson TD, Callaghan JT, Einolf HJ, Fischer V, Gan L, Grimm S, et al. The conduct of in vitro and in vivo drug-drug interaction studies: a PhRMA perspective. J Clin Pharmacol. 2003;43(5):443–69. https://doi.org/10.1177/0091270003252519.

    Article  CAS  PubMed  Google Scholar 

  4. Mao J, Mohutsky MA, Harrelson JP, Wrighton SA, Hall SD. Prediction of CYP3A-mediated drug-drug interactions using human hepatocytes suspended in human plasma. Drug Metab Dispos. 2011;39(4):591 LP–602.

    Article  Google Scholar 

  5. Xu L, Chen Y, Pan Y, Skiles GL, Shou M. Prediction of human drug-drug interactions from time-dependent inactivation of CYP3A4 in primary hepatocytes using a population-based simulator. Drug Metab Dispos. 2009;37(12):2330 LP–2339.

    Article  Google Scholar 

  6. Feng BY, Simeonov A, Jadhav A, Babaoglu K, Inglese J, Shoichet BK, et al. A high-throughput screen for aggregation-based inhibition in a large compound library. J Med Chem. 2007;50(10):2385–90. https://doi.org/10.1021/jm061317y.

    Article  CAS  PubMed  Google Scholar 

  7. McGovern SL, Caselli E, Grigorieff N, Shoichet BK. A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. J Med Chem. 2002;45(8):1712–22. https://doi.org/10.1021/jm010533y.

    Article  CAS  PubMed  Google Scholar 

  8. McGovern SL, Helfand BT, Feng B, Shoichet BK. A specific mechanism of nonspecific inhibition. J Med Chem. 2003;46(20):4265–72. https://doi.org/10.1021/jm030266r.

    Article  CAS  PubMed  Google Scholar 

  9. Seidler J, McGovern SL, Doman TN, Shoichet BK. Identification and prediction of promiscuous aggregating inhibitors among known drugs. J Med Chem. 2003;46(21):4477–86. https://doi.org/10.1021/jm030191r.

    Article  CAS  PubMed  Google Scholar 

  10. Feng BY, Shelat A, Doman TN, Guy RK, Shoichet BK. High-throughput assays for promiscuous inhibitors. Nat Chem Biol. 2005;1:146.

    Article  CAS  Google Scholar 

  11. Feng BY, Shoichet BK. A detergent-based assay for the detection of promiscuous inhibitors. Nat Protoc. 2006;1(2):550–3. https://doi.org/10.1038/nprot.2006.77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Coan KED, Shoichet BK. Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors. J Am Chem Soc. 2008;130(29):9606–12. https://doi.org/10.1021/ja802977h.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Doak AK, Wille H, Prusiner SB, Shoichet BK. Colloid formation by drugs in simulated intestinal fluid. J Med Chem. 2010;53(10):4259–65. https://doi.org/10.1021/jm100254w.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Coan KED, Maltby DA, Burlingame AL, Shoichet BK. Promiscuous aggregate-based inhibitors promote enzyme unfolding. J Med Chem. 2009;52(7):2067–75. https://doi.org/10.1021/jm801605r.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sassano MF, Doak AK, Roth BL, Shoichet BK. Colloidal aggregation causes inhibition of G protein-coupled receptors. J Med Chem. 2013;56(6):2406–14. https://doi.org/10.1021/jm301749y.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Owen SC, Doak AK, Wassam P, Shoichet MS, Shoichet BK. Colloidal aggregation affects the efficacy of anticancer drugs in cell culture. ACS Chem Biol. 2012;7:1429–35.

    Article  CAS  Google Scholar 

  17. Kirchmair J, Göller AH, Lang D, Kunze J, Testa B, Wilson ID, et al. Predicting drug metabolism: experiment and/or computation? Nat Rev Drug Discov. 2015;14:387.

    Article  CAS  Google Scholar 

  18. Trasi NS, Taylor LS. Thermodynamics of highly supersaturated aqueous solutions of poorly water-soluble drugs - impact of a second drug on the solution Phase behavior and implications for combination products. J Pharm Sci. 2015;104(8):2583–93. https://doi.org/10.1002/jps.24528.

    Article  CAS  PubMed  Google Scholar 

  19. Alhalaweh A, Bergström CAS, Taylor LS. Compromised in vitro dissolution and membrane transport of multidrug amorphous formulations. J Control Release. 2016;229:172–82. https://doi.org/10.1016/j.jconrel.2016.03.028.

    Article  CAS  PubMed  Google Scholar 

  20. Lindfors L, Forssén S, Skantze P, Skantze U, Zackrisson A, Olsson U. Amorphous drug nanosuspensions. 2. Experimental determination of bulk monomer concentrations. Langmuir. 2006;22(3):911–6. https://doi.org/10.1021/la052367t.

    Article  CAS  PubMed  Google Scholar 

  21. Raina SA, Alonzo DE, Zhang GGZ, Gao Y, Taylor LS. Using environment-sensitive fluorescent probes to characterize liquid-liquid phase separation in supersaturated solutions of poorly water soluble compounds. Pharm Res. 2015;32(11):3660–73. https://doi.org/10.1007/s11095-015-1725-z.

    Article  CAS  PubMed  Google Scholar 

  22. Emoto C, Iwasaki K. Approach to predict the contribution of cytochrome P450 enzymes to drug metabolism in the early drug-discovery stage: the effect of the expression of cytochrome B5 with recombinant P450 enzymes. Xenobiotica. 2007;37(9):986–99. https://doi.org/10.1080/00498250701620692.

    Article  CAS  PubMed  Google Scholar 

  23. Tres F, Hall SD, Mohutsky MA, Taylor LS. Monitoring the phase behavior of supersaturated solutions of poorly water-soluble drugs using fluorescence techniques. J Pharm Sci. 2018;107(1):94–102.

    Article  CAS  Google Scholar 

  24. Simpson NJ, Solid-Phase K. Extraction: principles, techniques, and applications. Marcel Dekker, Inc: New York; 2000.

    Book  Google Scholar 

  25. Baird JA, Van Eerdenbrugh B, Taylor LS. A classification system to assess the crystallization tendency of organic molecules from undercooled melts. J Pharm Sci. 2010;99(9):3787–806. https://doi.org/10.1002/jps.22197.

    Article  CAS  PubMed  Google Scholar 

  26. Loftsson T, Vogensen SB, Desbos C, Jansook P. Carvedilol: solubilization and cyclodextrin complexation: a technical note. AAPS PharmSciTech. 2008;9(2):425–30. https://doi.org/10.1208/s12249-008-9055-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Karjalainen MJ, Neuvonen PJ, Backman JT. In vitro inhibition of CYP1A2 by model inhibitors, anti-inflammatory analgesics and female sex steroids: predictability of in vivo interactions. Basic Clin Pharmacol Toxicol. 2008;103(2):157–65. https://doi.org/10.1111/j.1742-7843.2008.00252.x.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors wish to acknowledge Eli Lilly and Co. for funding through the LRAP program.

Author information

Authors and Affiliations

  1. Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, West Lafayette, Indiana, 47907, USA

    Francesco Tres & Lynne S. Taylor

  2. Drug Disposition, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana, 46285, USA

    Maria M. Posada, Stephen D. Hall & Michael A. Mohutsky

Authors
  1. Francesco Tres
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria M. Posada
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stephen D. Hall
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael A. Mohutsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lynne S. Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lynne S. Taylor.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tres, F., Posada, M.M., Hall, S.D. et al. The Effect of Promiscuous Aggregation on in Vitro Drug Metabolism Assays. Pharm Res 36, 170 (2019). https://doi.org/10.1007/s11095-019-2713-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11095-019-2713-5

KEY WORDS

Search

Navigation