Quantification of drug metabolising enzymes and transporter proteins in the paediatric duodenum via LC-MS/MS proteomics using a QconCAT technique

Jan Goelen; Gillian Farrell; Jonathan McGeehan; Christopher M Titman; Nicholas J W Rattray; Trevor N Johnson; Richard D Horniblow; Hannah K Batchelor

doi:10.1016/j.ejpb.2023.08.011

Quantification of drug metabolising enzymes and transporter proteins in the paediatric duodenum via LC-MS/MS proteomics using a QconCAT technique

Jan Goelen, Gillian Farrell, Jonathan McGeehan, Christopher M Titman, Nicholas J W Rattray, Trevor N Johnson, Richard D Horniblow, Hannah K Batchelor^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

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Abstract

Characterising the small intestine absorptive membrane is essential to enable prediction of the systemic exposure of oral formulations. In particular, the ontogeny of key intestinal Drug Metabolising Enzymes and Transporter (DMET) proteins involved in drug disposition needs to be elucidated to allow for accurate prediction of the PK profile of drugs in the paediatric cohort.

Using pinch biopsies from the paediatric duodenum (n=36; aged 11 months to 15 years), the abundance of 21 DMET proteins and two enterocyte markers were quantified via LC-MS/MS. An established LCMS nanoflow method was translated to enable analysis on a microflow LC system, and a new stable-isotope-labelled QconCAT standard developed to enable quantification of these proteins. Villin-1 was used to standardise abundancy values. The observed abundancies and ontogeny profiles, agreed with adult LC-MS/MS-based data, and historic paediatric data obtained via western blotting. A linear trend with age was observed for duodenal CYP3A4 and CES2 only. As this work quantified peptides on a pinch biopsy coupled with a microflow method, future studies using a wider population range are very feasible. Furthermore, this DMET ontogeny data can be used to inform paediatric PBPK modelling and to enhance the understanding of oral drug absorption and gut bioavailability in paediatric populations.

Original language	English
Pages (from-to)	68-77
Number of pages	10
Journal	European Journal of Pharmaceutics and Biopharmaceutics
Volume	191
Early online date	23 Aug 2023
DOIs	https://doi.org/10.1016/j.ejpb.2023.08.011
Publication status	Published - Oct 2023

Bibliographical note

Acknowledgments:
Certara funded this work as a PhD project for J. G.. The authors thank Gopal Pawar for his advice regarding isolation methods. The authors also thank PolyQuant and Florian Christoph Sigloch for their contribution in synthesising the PaedCAT. The authors would like to thank the Strathclyde Centre of Molecular Bioscience for access to instrumentation and Shimazu-UK for advice on method development.

Copyright:
© 2023 The Authors. Published by Elsevier B.V.

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Goelen, J., Farrell, G., McGeehan, J., Titman, C. M., J W Rattray, N., Johnson, T. N., Horniblow, R. D., & Batchelor, H. K. (2023). Quantification of drug metabolising enzymes and transporter proteins in the paediatric duodenum via LC-MS/MS proteomics using a QconCAT technique. European Journal of Pharmaceutics and Biopharmaceutics, 191, 68-77. https://doi.org/10.1016/j.ejpb.2023.08.011

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title = "Quantification of drug metabolising enzymes and transporter proteins in the paediatric duodenum via LC-MS/MS proteomics using a QconCAT technique",

abstract = "Characterising the small intestine absorptive membrane is essential to enable prediction of the systemic exposure of oral formulations. In particular, the ontogeny of key intestinal Drug Metabolising Enzymes and Transporter (DMET) proteins involved in drug disposition needs to be elucidated to allow for accurate prediction of the PK profile of drugs in the paediatric cohort. Using pinch biopsies from the paediatric duodenum (n=36; aged 11 months to 15 years), the abundance of 21 DMET proteins and two enterocyte markers were quantified via LC-MS/MS. An established LCMS nanoflow method was translated to enable analysis on a microflow LC system, and a new stable-isotope-labelled QconCAT standard developed to enable quantification of these proteins. Villin-1 was used to standardise abundancy values. The observed abundancies and ontogeny profiles, agreed with adult LC-MS/MS-based data, and historic paediatric data obtained via western blotting. A linear trend with age was observed for duodenal CYP3A4 and CES2 only. As this work quantified peptides on a pinch biopsy coupled with a microflow method, future studies using a wider population range are very feasible. Furthermore, this DMET ontogeny data can be used to inform paediatric PBPK modelling and to enhance the understanding of oral drug absorption and gut bioavailability in paediatric populations.",

author = "Jan Goelen and Gillian Farrell and Jonathan McGeehan and Titman, {Christopher M} and {J W Rattray}, Nicholas and Johnson, {Trevor N} and Horniblow, {Richard D} and Batchelor, {Hannah K}",

note = "Acknowledgments: Certara funded this work as a PhD project for J. G.. The authors thank Gopal Pawar for his advice regarding isolation methods. The authors also thank PolyQuant and Florian Christoph Sigloch for their contribution in synthesising the PaedCAT. The authors would like to thank the Strathclyde Centre of Molecular Bioscience for access to instrumentation and Shimazu-UK for advice on method development. Copyright: {\textcopyright} 2023 The Authors. Published by Elsevier B.V.",

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Goelen, J, Farrell, G, McGeehan, J, Titman, CM, J W Rattray, N, Johnson, TN, Horniblow, RD & Batchelor, HK 2023, 'Quantification of drug metabolising enzymes and transporter proteins in the paediatric duodenum via LC-MS/MS proteomics using a QconCAT technique', European Journal of Pharmaceutics and Biopharmaceutics, vol. 191, pp. 68-77. https://doi.org/10.1016/j.ejpb.2023.08.011

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AU - J W Rattray, Nicholas

AU - Johnson, Trevor N

AU - Horniblow, Richard D

AU - Batchelor, Hannah K

N1 - Acknowledgments: Certara funded this work as a PhD project for J. G.. The authors thank Gopal Pawar for his advice regarding isolation methods. The authors also thank PolyQuant and Florian Christoph Sigloch for their contribution in synthesising the PaedCAT. The authors would like to thank the Strathclyde Centre of Molecular Bioscience for access to instrumentation and Shimazu-UK for advice on method development. Copyright: © 2023 The Authors. Published by Elsevier B.V.

PY - 2023/10

Y1 - 2023/10

N2 - Characterising the small intestine absorptive membrane is essential to enable prediction of the systemic exposure of oral formulations. In particular, the ontogeny of key intestinal Drug Metabolising Enzymes and Transporter (DMET) proteins involved in drug disposition needs to be elucidated to allow for accurate prediction of the PK profile of drugs in the paediatric cohort. Using pinch biopsies from the paediatric duodenum (n=36; aged 11 months to 15 years), the abundance of 21 DMET proteins and two enterocyte markers were quantified via LC-MS/MS. An established LCMS nanoflow method was translated to enable analysis on a microflow LC system, and a new stable-isotope-labelled QconCAT standard developed to enable quantification of these proteins. Villin-1 was used to standardise abundancy values. The observed abundancies and ontogeny profiles, agreed with adult LC-MS/MS-based data, and historic paediatric data obtained via western blotting. A linear trend with age was observed for duodenal CYP3A4 and CES2 only. As this work quantified peptides on a pinch biopsy coupled with a microflow method, future studies using a wider population range are very feasible. Furthermore, this DMET ontogeny data can be used to inform paediatric PBPK modelling and to enhance the understanding of oral drug absorption and gut bioavailability in paediatric populations.

AB - Characterising the small intestine absorptive membrane is essential to enable prediction of the systemic exposure of oral formulations. In particular, the ontogeny of key intestinal Drug Metabolising Enzymes and Transporter (DMET) proteins involved in drug disposition needs to be elucidated to allow for accurate prediction of the PK profile of drugs in the paediatric cohort. Using pinch biopsies from the paediatric duodenum (n=36; aged 11 months to 15 years), the abundance of 21 DMET proteins and two enterocyte markers were quantified via LC-MS/MS. An established LCMS nanoflow method was translated to enable analysis on a microflow LC system, and a new stable-isotope-labelled QconCAT standard developed to enable quantification of these proteins. Villin-1 was used to standardise abundancy values. The observed abundancies and ontogeny profiles, agreed with adult LC-MS/MS-based data, and historic paediatric data obtained via western blotting. A linear trend with age was observed for duodenal CYP3A4 and CES2 only. As this work quantified peptides on a pinch biopsy coupled with a microflow method, future studies using a wider population range are very feasible. Furthermore, this DMET ontogeny data can be used to inform paediatric PBPK modelling and to enhance the understanding of oral drug absorption and gut bioavailability in paediatric populations.

U2 - 10.1016/j.ejpb.2023.08.011

DO - 10.1016/j.ejpb.2023.08.011

M3 - Article

C2 - 37625656

SN - 0939-6411

VL - 191

SP - 68

EP - 77

JO - European Journal of Pharmaceutics and Biopharmaceutics

JF - European Journal of Pharmaceutics and Biopharmaceutics

ER -

Quantification of drug metabolising enzymes and transporter proteins in the paediatric duodenum via LC-MS/MS proteomics using a QconCAT technique

Abstract

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