Christine Wegler

Variability in Mass Spectrometry-based Quantification of Clinically Relevant Drug Transporters and Drug Metabolizing Enzymes

Many different methods are used for mass-spectrometry-based protein quantification in pharmacokinetics and systems pharmacology. It has not been established to what extent the results from these various methods are comparable. Here, we compared six different mass spectrometry-based proteomics methods by measuring the expression of clinically relevant drug transporters and metabolizing enzymes in human liver. Mean protein concentrations were in general quantified to similar levels by methods using whole tissue lysates. Methods using subcellular membrane fractionation gave incomplete enrichment of the proteins. When the enriched proteins were adjusted to levels in whole tissue lysates, they were on average 4-fold lower than those quantified directly in whole tissue lysates. The differences in protein levels were propagated into differences in predictions of hepatic clearance. In conclusion, caution is needed when comparing and applying quantitative proteomics data obtained with different methods, especially since membrane fractionation is common practice for protein quantification used in drug clearance predictions.

Intracellular drug bioavailability: a new predictor of system dependent drug disposition

Intracellular drug exposure is influenced by cell- and tissue-dependent expression of drug-transporting proteins and metabolizing enzymes. Here, we introduce the concept of intracellular bioavailability (Fic) as the fraction of extracellular drug available to bind intracellular targets, and we assess how Fic is affected by cellular drug disposition processes. We first investigated the impact of two essential drug transporters separately, one influx transporter (OATP1B1; SLCO1B1) and one efflux transporter (P-gp; ABCB1), in cells overexpressing these proteins. We showed that OATP1B1 increased Fic of its substrates, while P-gp decreased Fic. We then investigated the impact of the concerted action of multiple transporters and metabolizing enzymes in freshly-isolated human hepatocytes in culture configurations with different levels of expression and activity of these proteins. We observed that Fic was up to 35-fold lower in the configuration with high expression of drug-eliminating transporters and enzymes. We conclude that Fic provides a measurement of the net impact of all cellular drug disposition processes on intracellular bioavailable drug levels. Importantly, no prior knowledge of the involved drug distribution pathways is required, allowing for high-throughput determination of drug access to intracellular targets in highly defined cell systems (e.g., single-transporter transfectants) or in complex ones (including primary human cells).

Effects of Osteoporosis-Inducing Drugs on Vitamin D-Related Gene Transcription and Mineralization in MG-63 and Saos-2 Cells.

Vitamin D3 is important for calcium and phosphate homeostasis. To exert its effects, vitamin D3 has to be enzymatically activated into 1,25D3 (1,25‐dihydroxyvitamin D3). Regulation by endogenous vitamin D metabolites of the activation and inactivation of 1,25D3 is important to maintain adequate amounts of active vitamin D3. Vitamin D deficiency and low bone mineral density have been linked to treatments with antiretroviral drugs and glucocorticoids. However, the causes of drug‐induced osteoporosis remain unclear. The antiretroviral drugs efavirenz and ritonavir as well as the glucocorticoid dexamethasone were included in this study. Their effects on transcription of vitamin D‐regulating enzymes in MG‐63 cells were investigated. Ritonavir and dexamethasone both induced transcription of CYP27B1, the enzyme responsible for the formation of 1,25D3. Efavirenz, however, suppressed CYP27B1 expression. When administered together with endogenous vitamin D metabolites, dexamethasone and efavirenz counteracted the 1,25D3‐mediated up‐regulation of CYP24A1, which inactivates 1,25D3. This suggests that the drugs may interfere with local regulation of the vitamin D metabolizing system in osteoblasts. Studies on mineralization were performed in MG‐63 cells and Saos‐2 cells by measuring calcium concentrations accumulated over time. The effects of efavirenz, ritonavir and dexamethasone and/or vitamin D metabolites were examined. 1,25D3 induced mineralization in both cell lines. Efavirenz administered alone did not affect mineralization but suppressed the inducing effects of 1,25D3 on mineralization in both MG‐63 cells and Saos‐2 cells. In summary, the results suggest that antiretroviral drugs and glucocorticoids may adversely affect bone by interference with the vitamin D system in osteoblasts.

Direct Quantification of Cytochromes P450 and Drug Transporters—A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates

The quantification of drug metabolizing enzymes and transporters has recently been revolutionized on the basis of targeted proteomic approaches. Isotope-labeled peptides are used as standards for the quantification of the corresponding proteins in enzymatically fragmented samples. However, hurdles in these approaches are low throughput and tedious sample prefractionation steps prior to mass spectrometry (MS) readout. We have developed an assay platform using sensitive and selective immunoprecipitation coupled with mass spectrometric readout allowing the quantification of proteins directly from whole cell lysates using less than 20,000 cells per analysis. Peptide group-specific antibodies (triple X proteomics antibodies) enable the enrichment of proteotypic peptides sharing a common terminus. These antibodies were employed to establish a MS-based immunoassay panel for the quantification of 14 cytochrome P450 (P450) enzymes and nine transporters. We analyzed the P450 enzyme and transporter levels in genotyped liver tissue homogenates and microsomes, and in samples from a time course induction experiment in human hepatocytes addressing different induction pathways. For the analysis of P450 enzymes and transporters only a minute amount of sample is required and no prefractionation is necessary, thus the assay platform bears the potential to bridge cell culture model experiments and results from whole organ tissue studies.