Quantitative Targeted Absolute Proteomics-Based Adme Research as A New Path to Drug Discovery and Development: Methodology, Advantages, Strategy, and Prospects

Ohtsuki, Sumio; Uchida, Yasuo; Kubo, Yoshiyuki; Terasaki, Tetsuya

doi:10.1002/jps.22612

Cited by 126 publications

(97 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…MRM analysis of diagnostic peptides with stable isotope-labeled internal standards and reference peptides allows for sensitive and selective absolute quantification of protein concentrations with a linear dynamic range similar to that of small molecules (Elliott et al, 2009;Ohtsuki et al, 2011). Integral membrane proteins yet pose an analytical challenge due to their hydrophobic nature and low abundance, as seen for many drug transporters (Barnidge et al, 2003;Kamiie et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

Absolute Quantification and Differential Expression of Drug Transporters, Cytochrome P450 Enzymes, and UDP-Glucuronosyltransferases in Cultured Primary Human Hepatocytes

Schaefer¹,

Ohtsuki²,

Kawakami³

et al. 2011

Drug Metab Dispos

Self Cite

123

View full text Add to dashboard Cite

ABSTRACT:The levels of metabolizing enzymes and transporters expressed in hepatocytes are decisive factors for hepatobiliary disposition of most drugs. Induction via nuclear receptor activation can significantly alter those levels, with the coregulation of multiple enzymes and transporters occurring to different extents. Here, we report the use of a targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for concurrent quantification of multiple cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and transporter proteins in cultured primary human hepatocytes. The effects of culture format (i.e., sandwich culture versus conventional culture) and of dexamethasone (DEX) media concentrations on mRNA, protein, and activity levels were determined for three donors, and protein expression was compared with that in liver. In general, P450 and UGT expression was lower in hepatocyte cultures than that in liver, and CYP2C9 was found to be the most abundant P450 isoform expressed in cultured hepatocytes. The sandwich culture format and 0.1 M DEX in media retained the protein expression in the hepatocytes closest to the levels found in liver. However, higher in vitro expression was observed for drug transporters, especially for multidrug resistance protein 1 and breast cancer resistance protein. Direct protein quantification was applied successfully to study in vitro induction in sandwich cultured primary hepatocytes in a 24-well format using the prototypical inducers rifampicin, omeprazole, and phenobarbital. We conclude that targeted absolute LC-MS/MS quantification of drugmetabolizing enzymes and transporters can broaden the scope and significantly increase the impact of in vitro drug metabolism studies, such as induction, as an important supplement or future alternative to mRNA and activity data.

show abstract

Section: Discussionmentioning

confidence: 99%

Absolute Quantification and Differential Expression of Drug Transporters, Cytochrome P450 Enzymes, and UDP-Glucuronosyltransferases in Cultured Primary Human Hepatocytes

Schaefer¹,

Ohtsuki²,

Kawakami³

et al. 2011

Drug Metab Dispos

Self Cite

123

View full text Add to dashboard Cite

show abstract

“…Understanding the difference of membrane protein expression is the key to bridge the gap between in vitro models and interspecies that underlie interexperimental variation and to extrapolate in vitro data to in vivo (5,6,18,46,47,75). Appreciation of interspecies differences of proteins involved in drug elimination and their expression levels can increase the confidence in data interpretation.…”

Section: Examples Of Quantitative Targeted Proteomics For Membrane Trmentioning

confidence: 99%

Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

Qiu

Zhang

Lai

2014

AAPS J

View full text Add to dashboard Cite

Abstract. Although global proteomics has shown promise for discovery of many new proteins, biomarkers, protein modifications, and polymorphisms, targeted proteomics is emerging in the proteomics research field as a complement to untargeted shotgun proteomics, particularly when a determined set of low-abundance functional proteins need to be measured. The function and expression of proteins related to drug absorption, distribution, metabolism, and excretion (ADME) such as cytochrome P450 enzymes and membrane transporters are of great interest in biopharmaceutical research. Since the variation in ADME-related protein expression is known to be a major complicating factor encountered during in vitro-in vivo and in vivo-in vivo extrapolations (IVIVE), the accurate quantification of the ADME proteins in complex biological systems becomes a fundamental element in establishing IVIVE for pharmacokinetic predictions. In this review, we provide an overview of relevant methodologies followed by a summary of recent applications encompassing mass spectrometry-based targeted quantifications of membrane transporters.KEY WORDS: drug absorption, distribution, metabolism, and excretion (ADME); drug transporters; in vitro-in vivo and in vivo-in vivo extrapolations (IVIVE); LC-MS/MS; quantitative targeted proteomics.

show abstract

“…The target peptide for quantification was selected based on in silico selection criteria as described previously (Kamiie et al, 2008. Quantification of human transporters and other membrane proteins was based on the MRM conditions reported previously (Kamiie et al, 2008;Ohtsuki et al, 2011;Sakamoto et al, 2011;Shawahna et al, 2011;Uchida et al, 2011). The peptide sequence for NADPH-P450 reductase (P450R) was FAVFGLGNK, and L was labeled with stable isotope.…”

Section: Methodsmentioning

confidence: 99%

Simultaneous Absolute Protein Quantification of Transporters, Cytochromes P450, and UDP-Glucuronosyltransferases as a Novel Approach for the Characterization of Individual Human Liver: Comparison with mRNA Levels and Activities

et al. 2011

Self Cite

View full text Add to dashboard Cite

ABSTRACT:The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDPglucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.

show abstract

Quantitative Targeted Absolute Proteomics-Based Adme Research as A New Path to Drug Discovery and Development: Methodology, Advantages, Strategy, and Prospects

Cited by 126 publications

References 46 publications

Absolute Quantification and Differential Expression of Drug Transporters, Cytochrome P450 Enzymes, and UDP-Glucuronosyltransferases in Cultured Primary Human Hepatocytes

Absolute Quantification and Differential Expression of Drug Transporters, Cytochrome P450 Enzymes, and UDP-Glucuronosyltransferases in Cultured Primary Human Hepatocytes

Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

Simultaneous Absolute Protein Quantification of Transporters, Cytochromes P450, and UDP-Glucuronosyltransferases as a Novel Approach for the Characterization of Individual Human Liver: Comparison with mRNA Levels and Activities

Contact Info

Product

Resources

About