Rats are extensively used as a preclinical model for
assessing
drug pharmacokinetics (PK) and tissue distribution; however, successful
translation of the rat data requires information on the differences
in drug metabolism and transport mechanisms between rats and humans.
To partly fill this knowledge gap, we quantified clinically relevant
drug-metabolizing enzymes and transporters (DMETs) in the liver and
different intestinal segments of Sprague-Dawley rats. The levels of
DMET proteins in rats were quantified using the global proteomics-based
total protein approach (TPA) and targeted proteomics. The abundance
of the major DMET proteins was largely comparable using quantitative
global and targeted proteomics. However, global proteomics-based TPA
was able to detect and quantify a comprehensive list of 66 DMET proteins
in the liver and 37 DMET proteins in the intestinal segments of SD
rats without the need for peptide standards. Cytochrome P450 (Cyp)
and UDP-glycosyltransferase (Ugt) enzymes were mainly detected in
the liver with the abundance ranging from 8 to 6502 and 74 to 2558
pmol/g tissue. P-gp abundance was higher in the intestine (124.1 pmol/g)
as compared to that in the liver (26.6 pmol/g) using the targeted
analysis. Breast cancer resistance protein (Bcrp) was most abundant
in the intestinal segments, whereas organic anion transporting polypeptides
(Oatp) 1a1, 1a4, 1b2, and 2a1 and multidrug resistance proteins (Mrp)
2 and 6 were predominantly detected in the liver. To demonstrate the
utility of these data, we modeled digoxin PK by integrating protein
abundance of P-gp and Cyp3a2 into a physiologically based PK (PBPK)
model constructed using PK-Sim software. The model was able to reliably
predict the systemic as well as tissue concentrations of digoxin in
rats. These findings suggest that proteomics-informed PBPK models
in preclinical species can allow mechanistic PK predictions in animal
models including tissue drug concentrations.
Aiming to elucidate the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) on drug metabolism, we explored the array of its protein-protein interactions (interactome) in human liver microsomes (HLM) with chemical crosslinking mass spectrometry (CXMS). Our strategy employs membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide and 4-(N-succinimidylcarboxy)benzophenone. Exposure of bait-incorporated HLM samples to light was followed by isolating the His-tagged bait protein and its crosslinked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the crosslinked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively crosslinked partners of CYP2E1 are the cytochromes P450 2A6, 2C8, 3A4, 4A11, and 4F2, UDP-glucuronosyltransferases (UGTs) 1A and 2B, fatty aldehyde dehydrogenase (ALDH3A2), epoxide hydrolase 1 (EPHX1), disulfide oxidase 1α (ERO1L), and ribophorin II (RPN2). These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes.
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