Targeted LC-MS/MS Proteomics-Based Strategy To Characterize in Vitro Models Used in Drug Metabolism and Transport Studies.

Subcellular fractionation of tissue homogenate provides enriched in vitro models (e.g., microsomes, cytosol, or membranes), which are routinely used in the drug metabolism or transporter activity and protein abundance studies. However, batch-to-batch or interlaboratory variability in the recovery, enrichment, and purity of the subcellular fractions can affect performance of in vitro models leading to inaccurate in vitro to in vivo extrapolation (IVIVE) of drug clearance. To evaluate the quality of subcellular fractions, we developed a simple, targeted, and sensitive LC-MS/MS proteomics-based strategy, which relies on determination of protein markers of various cellular organelles, i.e., plasma membrane, cytosol, nuclei, mitochondria, endoplasmic reticulum (ER), lysosomes, peroxisomes, cytoskeleton, and exosomes. Application of the quantitative proteomics method confirmed a significant effect of processing variables (i.e., homogenization method and centrifugation speed) on the recovery, enrichment, and purity of isolated proteins in microsomes and cytosol. Particularly, markers of endoplasmic reticulum lumen and mitochondrial lumen were enriched in the cytosolic fractions as a result of their release during homogenization. Similarly, the relative recovery and composition of the total membrane fraction isolated from cell vs tissue samples was quantitatively different and should be considered in IVIVE. Further, analysis of exosomes isolated from sandwich-cultured hepatocyte media showed the effect of culture duration on compositions of purified exosomes. Therefore, the quantitative proteomics-based strategy developed here can be applied for efficient and simultaneous determination of multiple protein markers of various cellular organelles when compared to antibody- or activity-based assays and can be used for quality control of subcellular fractionation procedures including in vitro model development for drug metabolism and transport studies.

The Promises of Quantitative Proteomics in Precision Medicine.

Precision medicine approach has a potential to ensure optimum efficacy and safety of drugs at individual patient level. Physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models could play a significant role in precision medicine by predicting interindividual variability in drug disposition and response. In order to develop robust PBPK/PD models, it is imperative that the critical physiological parameters affecting drug disposition and response and their variability are precisely characterized. Currently used PBPK/PD modeling software, for example, Simcyp and Gastroplus, encompass information such as organ volumes, blood flows to organs, body fat composition, glomerular filtration rate, etc. However, the information on the interindividual variability of the majority of the proteins associated with PK and PD, for example, drug metabolizing enzymes, transporters, and receptors, are not fully incorporated into these PBPK modeling platforms. Such information is significant because the population factors such as age, genotype, disease, and gender can affect abundance or activity of these proteins. To fill this critical knowledge gap, mass spectrometry-based quantitative proteomics has emerged as an important technique to characterize interindividual variability in the protein abundance of drug metabolizing enzymes, transporters, and receptors. Integration of these quantitative proteomics data into in silico PBPK/PD modeling tools will be crucial toward precision medicine.

Age-Dependent Absolute Abundance of Hepatic Carboxylesterases (CES1 and CES2) by LC-MS/MS Proteomics: Application to PBPK Modeling of Oseltamivir In Vivo Pharmacokinetics in Infants.

The age-dependent absolute protein abundance of carboxylesterase (CES) 1 and CES2 in human liver was investigated and applied to predict infant pharmacokinetics (PK) of oseltamivir. The CES absolute protein abundance was determined by liquid chromatography-tandem mass spectrometry proteomics in human liver microsomal and cytosolic fractions prepared from tissue samples obtained from 136 pediatric donors and 35 adult donors. Two surrogate peptides per protein were selected for the quantification of CES1 and CES2 protein abundance. Purified CES1 and CES2 protein standards were used as calibrators, and the heavy labeled peptides were used as the internal standards. In hepatic microsomes, CES1 and CES2 abundance (in picomoles per milligram total protein) increased approximately 5-fold (315.2 vs. 1664.4) and approximately 3-fold (59.8 vs. 174.1) from neonates to adults, respectively. CES1 protein abundance in liver cytosol also showed age-dependent maturation. Oseltamivir carboxylase activity was correlated with protein abundance in pediatric and adult liver microsomes. The protein abundance data were then used to model in vivo PK of oseltamivir in infants using pediatric physiologically based PK modeling and incorporating the protein abundance-based ontogeny function into the existing pediatric Simcyp model. The predicted pediatric area under the curve, maximal plasma concentration, and time for maximal plasma concentration values were below 2.1-fold of the clinically observed values, respectively.

An Optimized Method for Protein Extraction from OCT-Embedded Human Kidney Tissue for Protein Quantification by LC-MS/MS Proteomics.

The existing biobanks of remnant tissue from clinically indicated kidney biopsies are attractive potential reservoirs for quantification of clinically relevant human tissue proteins by quantitative proteomics. However, a significant caveat of this strategy is that the tissues are often preserved in optimal cutting temperature (OCT) medium. Although OCT is an effective method of preserving the morphologic and immunohistological characteristics of tissues for later study, it significantly impacts efforts to quantify protein expression by liquid chromatography-tandem mass spectrometry methods. We report here a simple, reproducible, and cost-effective procedure to extract proteins from OCT-embedded tissue samples. Briefly, the excess frozen OCT medium was scraped before thawing from the tissue specimens stored at -80°C for ∼3 months. The tissue samples were homogenized and diethyl ether/methanol extraction was performed to remove the remaining OCT medium. The recovered protein was denatured, reduced, and alkylated. The second step of protein extraction and desalting was performed by chloroform/methanol/water extraction of denatured proteins. The resultant protein pellet was trypsin-digested and the marker proteins of various kidney cellular compartments were quantified by targeted selective reaction monitoring proteomics. Upon comparison of peptide signals from OCT-embedded tissue and flash-frozen tissue from the same donors, both individual protein quantities, and their interindividual variabilities, were similar. Therefore, the approach reported here can be applied to clinical reservoirs of OCT-preserved kidney tissue to be used for quantitative proteomics studies of clinically relevant proteins expressed in different parts of the kidney (including drug transporters and metabolizing enzymes).

Developmental Regulation of Drug-Processing Genes in Livers of Germ-Free Mice.

Very little is known about the effect of gut microbiota on the ontogeny of drug-processing genes (DPGs) in liver. In this study, livers were harvested from conventional (CV) and germ-free (GF) male and female mice from 1 to 90 days of age. RNA-Seq in livers of 90-day-old male mice showed that xenobiotic metabolism was the most downregulated pathway within the mRNA transcriptome in absence of intestinal bacteria. In male livers, the mRNAs of 67 critical DPGs partitioned into 4 developmental patterns (real-time-quantitative polymerase chain reaction): Pattern-1 gradually increased to adult levels in livers of CV mice and were downregulated in livers of GF mice, as exemplified by the major drug-metabolizing enzymes cytochrome 3a (Cyp3a) family, which are prototypical pregnane X receptor (PXR)-target genes. Genes in Pattern-2 include Cyp1a2 (aryl hydrocarbon receptor-target gene), Cyp2c family, and Cyp2e1, which were all upregulated mainly at 90 days of age; as well as the peroxisome proliferator-activated receptor α (PPARα)-target genes Cyp4a family and Aldh3a2, which were upregulated not only in 90-days adult age, but also between neonatal and adolescent ages (from 1 to 30 days of age). Genes in Pattern-3 were enriched predominantly in livers of 15-day-old mice, among which the sterol-efflux transporter dimers Abcg5/Abcg8 were downregulated in GF mice. Genes in Pattern-4 were neonatal-enriched, among which the transporter Octn1 mRNA tended to be lower in GF mice at younger ages but higher in adult GF mice as compared with age-matched CV mice. Protein assays confirmed the downregulation of the PXR-target gene Cyp3a protein (Western-blot and liquid chromatography tandem mass spectroscopy), and decreased Cyp3a enzyme activities in male GF livers. Increased microsomal-Cyp4a proteins and nuclear-PPARα were also observed in male GF livers. Interestingly, in contrast to male livers, the mRNAs of Cyp2c or Cyp4a were not readily upregulated in female GF livers approaching adult age, suggesting the maturation of female-specific hormones interferes with the interactions between intestinal microbiota and DPG ontogeny. In conclusion, intestinal microbiota markedly impacts the ontogeny of many hepatic DPGs in a gender-specific manner.