Skip to main content
SpringerLink
Log in

Rapidly Distinguishing Reversible and Time-Dependent CYP450 Inhibition Using Human Liver Microsomes, Co-incubation, and Continuous Fluorometric Kinetic Analyses

  • Protocol
  • First Online:
Optimization in Drug Discovery

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

  • 2627 Accesses

Abstract

In this chapter we have provided a step-by-step protocol of a 384-well plate fluorescence-based assay used for rapid identification of reversible and time-dependent CYP450 inhibition. This was accomplished by comparing the time-dependence pattern of IC50 values of potential test inhibitors using a co-incubation approach with continuous fluorometric kinetic measurements. Briefly, test compounds were mixed with NADPH and were serially diluted to eight different concentrations. The enzymatic reaction was initiated by adding a single recombinant CYP pre-mixed with its corresponding fluorescent substrate and a mixture of NADP+, G6P and MgCl2. The enzyme activity was measured every 2 min by fluorescence intensity (CYP product) over typically a 30-min time period. Inhibition percentages were calculated relative to controls that contained no inhibitors at each time point and IC50 values of inhibitors were calculated at different incubation time intervals. Plotting IC50 values vs. incubation time revealed three different patterns for test inhibitors that could be used to distinguish reversible and time-dependent inhibitors. IC50 values of reversible inhibitors either maintained within a narrow range, or increased with incubation time because of losing inhibitor as a result of metabolism or non-specific binding to the matrix. In contrast, IC50 values decreased with incubation time for time-dependent inhibitors because of irreversible reactions caused by progressive enzyme inactivation by reactive metabolite species generated during the incubation or other inactivation mechanisms. Results clearly suggest that this co-incubation in vitro continuous fluorometric kinetic assay using recombinant CYPs and fluorometric generating substrates is a valuable high-throughput assay for distinguishing reversible and time-dependent inhibitors for large compound collections.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Caldwell GW (2000) Compound optimization in early- and late-phase drug discovery: acceptable pharmacokinetic properties utilizing combined physicochemical, in vitro and in vivo screens. Curr Opin Drug Discov Devel 3:30–41

    PubMed  CAS  Google Scholar 

  2. Caldwell GW, Ritchie DM, Masucci JA, Hageman W, Yan Z (2001) The new pre-preclinical paradigm: compound optimization in early and late phase drug discovery. Curr Top Med Chem 1(5):353–366

    Article  PubMed  CAS  Google Scholar 

  3. Bjornsson TD, Callaghan JT, Einolf HJ, Fischer V, Gan L, Grimm S, John Kao S, King P, Miwa G, Ni L, Kumar G, McLeod J, Obach SR, Roberts S, Roe A, Shah A, Snikeris F, Sullivan JT, Tweedie D, Vega JM, Walsh J, Wrighton SA (2003) The conduct of in vitro and in vivo drug-drug interaction studies: a Pharmaceutical Research and Manufacturers of America (PhRMA). J Clin Pharmacol 43(5):443–469

    PubMed  CAS  Google Scholar 

  4. Caldwell GW, Yan Z, Tang W, Dasgupta M, Hasting B (2009) ADME optimization and toxicity assessment in early- and late-phase drug discovery. Curr Top Med Chem 9(11):965–980

    Article  PubMed  CAS  Google Scholar 

  5. Lin JH, Lu AYH (1998) Inhibition and induction of cytochrome P450 and the clinical implications. Clin Pharmacokinet 5(5):361–390

    Article  Google Scholar 

  6. Yan Z, Caldwell GW (2001) Metabolism profiling, and cytochrome P450 inhibition & induction in drug discovery. Curr Top Med Chem 1(5):403–425

    Article  PubMed  CAS  Google Scholar 

  7. Stresser DM (2004) High-through screening of human cytochrome P450 inhibitors using fluorometric substrates: methodology for 25 enzyme/substrate pairs. In: Yan Z, Caldwell GW (eds) Optimization in drug discovery: in vitro methods, Method in pharmacology and toxicology. Humana, Totowa, NJ

    Google Scholar 

  8. Yan Z, Caldwell GW (2004) Evaluation of cytochrome P450 inhibition in human liver microsome. In: Yan Z, Caldwell GW (eds) Optimization in drug discovery: in vitro methods, Method in pharmacology and toxicology. Humana, Totowa, NJ

    Chapter  Google Scholar 

  9. Riley RJ, Grime K, Weaver R (2007) Time-dependent CYP inhibition. Expert Opin Drug Metab Toxicol 3(1):51–66

    Article  PubMed  CAS  Google Scholar 

  10. Polasek T, Miner JO (2007) In vitro approaches to investigate mechanism-based inactivation of CYP enzymes. Expert Opin Drug Metab Toxicol 3(1):321–329

    Article  PubMed  CAS  Google Scholar 

  11. Yan Z, Caldwell GW (2012) The current status of time dependent CYP inhibition assay and in silico drug-drug interaction predictions. Curr Top Med Chem 12(11):1291–1297

    Article  PubMed  CAS  Google Scholar 

  12. Caldwell GW, Yan Z, Lang W, Masucci J (2012) The IC50 concept revisited. Curr Top Med Chem 12(11):1282–1290

    Article  PubMed  CAS  Google Scholar 

  13. Moody GC, Griffin SJ, Mather AN, McGinnity DF, Riley RJ (1998) Fully automated analysis of activities catalysed by the major human liver cytochrome P450 (CYP) enzymes: assessment of human CYP inhibition potential. Xenobiotica 29(1):53–75

    Article  Google Scholar 

  14. Mano Y, Usui T, Kamimura H (2007) Comparison of inhibition potentials of drugs against zidovudine glucuronidation in rat hepatocytes and liver microsomes. Drug Metab Dispos 35(4):602–606

    Article  PubMed  CAS  Google Scholar 

  15. Crespi CL, Stresser DM (2001) Fluorometric screening for metabolism-based drug-drug interactions. J Pharmacol Toxicol Methods 44(1):325–331

    Article  Google Scholar 

  16. Crespi CL, Miller VP, Penman BW (1997) Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450. Anal Biochem 248(1):188–190

    Article  PubMed  CAS  Google Scholar 

  17. Chu I, Favreau L, Soares T, Lin CC, Nomeir AA (2000) Validation of higher-throughput high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes. Rapid Commun Mass Spectrom 14(4):207–214

    Article  PubMed  CAS  Google Scholar 

  18. Bell L, Bickford S, Nguyen PH, Wang J, He T, Zhang B, Friche Y, Zimmerlin A, Urban L, Bojanic D (2008) Evaluation of fluorescence- and mass spectrometry-based CYP inhibition assays for use in drug discovery. J Biomol Screen 13(5):343–353

    Article  PubMed  CAS  Google Scholar 

  19. Poulin P, Kenny JR, Hop CECA, Haddad S (2012) In vitro-in vivo extrapolation of clearance: modeling hepatic metabolic clearance of highly bound drugs and comparative assessment with existing calculation methods. J Pharm Sci 101(2):838–851

    Article  PubMed  CAS  Google Scholar 

  20. Imai H, Kotegawa T, Ohashi K (2011) Duration of drug interactions: putative time courses after mechanism-based inhibition or induction of CYPs. Expert Rev Clin Pharmacol 4(4):409–411

    Article  PubMed  CAS  Google Scholar 

  21. Yan Z, Rafferty B, Caldwell GW, Masucci JA (2002) Rapidly distinguishing reversible and irreversible CYP450 inhibitors by using fluorometric kinetic analyses. Eur J Drug Metab Pharmacokinet 27(4):281–287

    Article  PubMed  CAS  Google Scholar 

  22. Yamamoto T, Suzuki A, Kohno Y (2002) Application of microtiter plate assay to evaluate inhibitory effects of various compounds on nine cytochrome P450 isoforms and to estimate their inhibition patterns. Drug Metab Pharmacokinet 17(5):437–448

    Article  PubMed  CAS  Google Scholar 

  23. Yamamoto T, Suzuki A, Kohno Y (2004) High-throughput screening for the assessment of time-dependent inhibitions of new drug candidates on recombinant CYP2D6 and CYP3A4 using a single concentration method. Xenobiotica 34(1):87–101

    Article  PubMed  CAS  Google Scholar 

  24. Naritomi Y, Teramura Y, Terashita S, Kagayama A (2004) Utility of microtiter plate assays for human cytochrome P450 inhibition studies in drug discovery: application of simple method for detecting quasi-irreversible and irreversible inhibitors. Drug Metab Pharmacokinet 19(1):55–61

    Article  PubMed  CAS  Google Scholar 

  25. Turpeinen M, Korhonen LE, Tolonen A, Uusitalo J, Juvonen R, Raunio H, Pelkonen O (2006) Cytochrome P450 (CYP) inhibition screening: comparison of three tests. Eur J Pharm Sci 29(2):130–138

    Article  PubMed  CAS  Google Scholar 

  26. Krippendorff BF, Neuhaus R, Lienau P, Reichel A, Huisinga W (2009) Mechanism-based inhibition: deriving KI and kinact directly from time-dependent IC50 values. J Biomol Screen 14(8):913–923

    Article  PubMed  CAS  Google Scholar 

  27. Sekiguchi N, Higashida A, Kato M, Nabuchi Y, Mitsui T, Takanashi K, Aso Y, Ishigai M (2009) Prediction of drug-drug interactions based on time-dependent inhibition from high throughput screening of cytochrome P450 3A4 inhibition. Drug Metab Pharmacokinet 24(6):500–510

    Article  PubMed  CAS  Google Scholar 

  28. Kajbaf M, Palmieri E, Longhi R, Fontana S (2010) Identifying a higher throughput assay for metabolism dependent inhibition (MDI). Drug Metab Lett 4(2):104–113

    Article  PubMed  CAS  Google Scholar 

  29. Salminen KA, Leppanen J, Venalainen JI, Pasanen M, Auriola S, Juvonen RO, Raunio H (2011) Simple, direct, and informative method for the assessment of CYP2C19 enzyme inactivation kinetics. Drug Metab Dispos 39(3):412–418

    Article  PubMed  CAS  Google Scholar 

  30. Watanabe A, Nakamura K, Okudaira N, Okazaki O, Sudo K (2007) Risk assessment for drug-drug interaction caused by metabolism-based inhibition of CYP3A using automated in vitro assay systems and its application in the early drug discovery process. Drug Metab Dispos 35(7):1232–1238

    Article  PubMed  CAS  Google Scholar 

  31. Berry LM, Zhao Z (2008) An examination of IC50 and IC50-shift experiments in assessing time-dependent inhibition of CYP3A4, CYP2D6 and CYP2C9 in human liver microsomes. Drug Metab Lett 2(1):51–59

    Article  PubMed  CAS  Google Scholar 

  32. Burt HJ, Galetin A, Houston JB (2010) IC50-based approaches as an alternative method for assessment of time-dependent inhibition of CYP3A4. Xenobiotica 40(5):331–343

    Article  PubMed  CAS  Google Scholar 

  33. Burt HJ, Pertinez H, Sall C, Collins C, Hyland R, Houston JB, Galetin A (2012) Progress curve mechanistic modeling approach for assessing time-dependent inhibition of CYP3A4. Drug Metab Dispos 40(9):1658–1667

    Article  PubMed  CAS  Google Scholar 

  34. Kenny JR, Mukadam S, Zhang C, Tay S, Collins C, Galetin A, Khojasteh SC (2012) Drug-drug interaction potential of marketed oncology drugs: in vitro assessment of time-dependent cytochrome P450 inhibition, reactive metabolite formation and drug-drug interaction prediction. Pharm Res 29(7):1960–1976

    Article  PubMed  CAS  Google Scholar 

  35. Chen Y, Liu L, Monshouwer M, Fretland AJ (2011) Determination of time-dependent inactivation of CYP3A4 in cryopreserved human hepatocytes and assessment of human drug-drug interactions. Drug Metab Dispos 39(11):2085–2092

    Article  PubMed  CAS  Google Scholar 

  36. Maeng HJ, Chow ECY, Fan J, Pang KS (2012) Physiologically based pharmacokinetic (PBPK) modeling: usefulness and applications. In: Lyubimov AV, Rodrigues AD, Sinz MA (eds) Encyclopedia of drug metabolism and interactions, vol 2. Wiley, New York, pp 637–684

    Google Scholar 

  37. Donato MT, Gomez-Lechon MJ (2006) Inhibition of P450 enzymes: an in vitro approach. Curr Enzym Inhib 2:281–304

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The views expressed here are solely those of the author and do not reflect the opinions of Janssen Research & Development, LLC.

Author information

Authors and Affiliations

  1. CREATe Analytical Sciences, Janssen Research & Development, LLC, Spring House, PA, USA

    Gary W. Caldwell & Zhengyin Yan

Authors
  1. Gary W. Caldwell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhengyin Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. CREATe Core Science Technologies, Janssen Research & Development, LLC, Spring House, Pennsylvania, USA

    Gary W. Caldwell

  2. CREATe Core Science Technologies, Janssen Research & Development, LLC, Spring House, Pennsylvania, USA

    Zhengyin Yan

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Caldwell, G.W., Yan, Z. (2014). Rapidly Distinguishing Reversible and Time-Dependent CYP450 Inhibition Using Human Liver Microsomes, Co-incubation, and Continuous Fluorometric Kinetic Analyses. In: Caldwell, G., Yan, Z. (eds) Optimization in Drug Discovery. Methods in Pharmacology and Toxicology. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-742-6_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-742-6_17

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-741-9

  • Online ISBN: 978-1-62703-742-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation