title: Lost in centrifugation: accounting for transporter protein losses in quantitative targeted absolute proteomics.
abstract: In drug development, considerable efforts are made to extrapolate from in vitro and preclinical findings to predict human drug disposition by using in vitro-in vivo extrapolation (IVIVE) approaches. Use of IVIVE strategies linked with physiologically based pharmacokinetic (PBPK) modeling is widespread, and regulatory agencies are accepting and occasionally requesting model analysis to support licensing submissions. Recently, there has been a drive to improve PBPK models by characterizing the absolute abundance of enzymes, transporters, and receptors within mammalian tissues and in vitro experimental systems using quantitative targeted absolute proteomics (QTAP). The absolute abundance of proteins relevant to processes governing drug disposition provided by QTAP will enable IVIVE-PBPK to incorporate terms for the abundance of enzymes and transporters in target populations. However, most studies that report absolute abundances of enzymes and transporter proteins do so in enriched membrane fractions so as to increase the abundance per sample, and thus the assay's sensitivity, rather than measuring the expected lower abundance in the more biologically meaningful whole cells or tissues. This communication discusses the balance between protein enrichment and potential loss during the preparation of membrane fractions from whole cells or tissues. Accounting for losses with recovery factors throughout the fractionation procedure provides a means to correct for procedural losses, thereby enabling the scaling of protein abundance from subcellular fractions to whole-cell or organ abundances. PBPK models based on corrected abundances will more closely resemble biological systems and facilitate development of more meaningful IVIVE scaling factors, producing more accurate quantitative predictions of drug disposition.
authors: Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford (M.D.H., G.W.), Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, (M.D.H., S.N., A.R-H.), and Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, Manchester (M.R.R., A.R-H.), United Kingdom matthew.harwood@certara.com.
authors: Matthew D
authors: MD
authors: Harwood
authors: Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford (M.D.H., G.W.), Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, (M.D.H., S.N., A.R-H.), and Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, Manchester (M.R.R., A.R-H.), United Kingdom.
authors: Matthew R
authors: MR
authors: Russell
authors: Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford (M.D.H., G.W.), Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, (M.D.H., S.N., A.R-H.), and Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, Manchester (M.R.R., A.R-H.), United Kingdom.
authors: Sibylle
authors: S
authors: Neuhoff
authors: Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford (M.D.H., G.W.), Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, (M.D.H., S.N., A.R-H.), and Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, Manchester (M.R.R., A.R-H.), United Kingdom.
authors: Geoffrey
authors: G
authors: Warhurst
authors: Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford (M.D.H., G.W.), Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, (M.D.H., S.N., A.R-H.), and Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, Manchester (M.R.R., A.R-H.), United Kingdom.
authors: Amin
authors: A
authors: Rostami-Hodjegan
mesh_terms: D000818:Animals; D002458:Cell Fractionation; D002498:Centrifugation; D006801:Humans; D026901:Membrane Transport Proteins / Q000032:analysis* / Q000302:isolation & purification; D008954:Models, Biological; D040901:Proteomics / Q000379:methods*
chemical_list: D026901:Membrane Transport Proteins