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Proteomic assessment of intestinal transporters
S12
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S1eS21
Increased ceramides also cause an elevation in fatty acid synthesis in liver which accounts for the NAFLD in in obese mice where FXR is constitutively activated. Decreasing ceramide levels through inhibition of intestinal FXR signaling is a feasible means to reduce metabolic diseases.
protein expression data to be presented in this project are hypothesis generating, which can be followed by the mechanistic studies to determine fundamental genetic or epigenetic mechanisms of age-dependent variability of expression of DMEs and transporters.
S38 QUANTITATIVE EXPRESSION OF ADME PROTEINS AT THE BLOOD-BRAIN BARRIER
S40 PROTEOMIC ASSESSMENT OF INTESTINAL TRANSPORTERS
Tetsuya Terasaki. Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Most of candidate compounds of CNS acting drug are substrate of the efflux transporter proteins such as P-gp/MDR1, BCRP and MRP4 expressing in the luminal membrane of the brain capillary endothelial cells, i.e., the bold-brain barrier (BBB). One of the most critical problems in the CNS acting drug discovery and development is that these efflux transport functions generate significantly lower unbound drug concentration in the brain than that of plasma. Therefore, it is a key issue to predict unbound drug concentration gradient between brain and plasma (Kp,uu) across the BBB. Our ultimate goal is to establish a novel strategy to solve this critical problem in the diseased human brain. Recently, we have succeeded to predict Kp,uu for several P-gp substrates in the normal brain of mouse and cynomolgus monkey based on the pharmacoprotemic model analysis combined with the quantitative analysis of P-gp protein expression in the BBB and the in vitro measurement of intrinsic P-gp efflux transport activities (J. Pharmacol. Exp. Ther.,339: 579 (2011), ibid 350: 578 (2014)) together with epileptic model mouse and chronically phenytoin administered normal mouse (Drug Metab Dispos. 42: 1719 (2014)). These results demonstrate that brain distributions of P-gp substrates could be predicted by assuming the in vivo intrinsic P-gp transport activity is the same to that of in vitro. Interestingly, Miller and colleagues have reported that P-gp transport activity is decreased by the treatment of inflammatory cytokines in a short period of time (Mol Pharmacol 69:462, 2006), suggesting that intrinsic P-gp activity is not always constant. Therefore, we have expanded our approach to the analysis of acute phase diseased model, i.e., oxidative stress model. Human brain capillary endothelial cell line (hCMEC/D3), an in vitro human BBB model, was treated with hydrogen peroxide for 20 min. Vinblastine efflux transport activities were significantly reduced. Based on phosphoproteomic analysis of D3 cells, candidate proteins regulating BBB P-gp transport function are identified. Combination of functional analysis and quantitative P-gp protein analysis revealed that caveolin-1 mediated internalization of P-gp protein into cytosolic space is involved in the apparent decrease of BBB P-gp efflux function, suggesting the localization of transporter protein in the brain capillary endothelial cells is also important subject for the deep understanding of disease effect on the BBB transport function. S39 QUANTITATIVE ANALYSIS OF HEPATIC TRANSPORTERS AND DMES DURING GROWTH AND DEVELOPMENT Bhagwat Prasad. University of Washington, Seattle, WA, USA The future of pediatric pharmacology lies on the better prediction of agedependent variability in drug disposition and response to ensure safety and efficacy of drugs in children. In this direction, physiologically based pharmacokinetic and pharmacodynamic models are proving to be promising. However, to establish physiological models, it is important that quantitative abundance data on drug metabolizing enzymes (DMEs) and transporters are obtained from pediatric vs. adult tissues. My presentation will discuss quantitative analysis of hepatic DMEs and transporters during growth and development. We used state-of-the-art quantitative proteomics technique to determine the effect of age on expression of multiple hepatic DMEs and transporters in 136 children and 38 adult subjects. Moreover, interplay of genetic polymorphism and ontogeny will be presented to reflect significance of genotyping studies together with ontogeny studies. An approach established in our laboratory to differentiate biological vs. technical variability, will be presented. These protein quantification data are critical for developing physiologically based pharmacokinetic models to predict drug disposition in children. The
Stefan Oswald. Center of Drug Absorption and Transport (C_DAT), University Medicine of Greifswald, Greifswald, Germany Transporter proteins are nowadays well recognized to affect the pharmacokinetics, efficacy and safety of many drugs in a significant manner. While there is a bunch of data available for hepatic uptake and biliary efflux transporters, the information on intestinal transporters is rather limited. Here, the research activities were mainly focused on ABCB1 (P-glycoprotein), which is known to limit the oral bioavailability of several drugs. However, little is known about the expression and function of other intestinal efflux as well as uptake transporters. The presentation will address the gene and protein expression of clinically relevant transporters along the entire human intestine. Moreover, their pharmacological relevance, the functional interplay of apical and basolateral transporters, their coordinate interaction with metabolizing enzymes as well as their transcriptional regulation will be discussed. S41 ABSTRACT UNAVAILABLE S42 PROTEO-METABOLOMICS IN ACETAMINOPHEN TOXICITY
ELUCIDATING
MECHANISMS
OF
Eric Chun Yong Chan, James Chun Yip Chan. National University of Singapore, Singapore, Singapore The adverse outcomes pathway (AOP) paradigm describes the events beginning from the initial interaction between a toxicant and its cellular target, which activates a cascade of cellular responses, culminating in cellular injury or death. However, the goal of populating the AOP remains elusive due to a lack of mechanistic understanding of the cellular targets and downstream events, which can include activation of signalling cascades, changes in protein post-translational modifications (PTMs), altered gene or protein expression levels, perturbations in organelle function and morphology, etc. In the absence of definitively identified toxicological targets, multi-omics approaches may provide mechanistic insight to pinpoint critical targets and perturbed pathways. The hepatotoxicant acetaminophen (APAP) has long been known to trigger covalent protein binding, yet these do not result in appreciable alterations in protein function that can be causally linked to the toxicity features elicited by APAP. Additionally, although many downstream events such as opening of the mitochondrial permeability transition pore (MPTP) and DNA fragmentation have been identified, the upstream triggers of these events are still unknown. Interestingly, secondary to overt covalent binding, APAP can covertly induce aberrant protein glutathionylation, which is a physiological PTM known to functionally modulate protein activity. To determine if APAP-induced protein glutathionylation is involved in APAP toxicity, we developed and applied the GluICAT proteomic methodology to globally profile aberrant glutathionylation temporally using human HepaRG cells. APAP was found to cause aberrant glutathionylation of critical proteins involved in energy metabolism, protein turnover, defense against cellular stress, calcium dynamics and mitochondrial permeability transition pore (MPTP) opening. A parallel metabolomics investigation permitted integrative proteo-metabolomics and revealed remarkable coherence in the functional deficits elicited by glutathionylation and corresponding metabolic derangements caused by APAP, with particular emphasis on energy metabolism (glycolysis, Krebs cycle, fatty acid b-oxidation and oxidative phosphorylation). Longitudinal analysis indicated these perturbations occurred within 3-6 h and preceded later events such as opening of the mitochondrial permeability transition pore, which suggests a causative, as opposed to an adaptive role. Separately, fibrate pre-treatment and Nacetylcysteine/S-adenosylmethionine supplementation is known to
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S1eS21
Increased ceramides also cause an elevation in fatty acid synthesis in liver which accounts for the NAFLD in in obese mice where FXR is constitutively activated. Decreasing ceramide levels through inhibition of intestinal FXR signaling is a feasible means to reduce metabolic diseases.
protein expression data to be presented in this project are hypothesis generating, which can be followed by the mechanistic studies to determine fundamental genetic or epigenetic mechanisms of age-dependent variability of expression of DMEs and transporters.
S38 QUANTITATIVE EXPRESSION OF ADME PROTEINS AT THE BLOOD-BRAIN BARRIER
S40 PROTEOMIC ASSESSMENT OF INTESTINAL TRANSPORTERS
Tetsuya Terasaki. Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Most of candidate compounds of CNS acting drug are substrate of the efflux transporter proteins such as P-gp/MDR1, BCRP and MRP4 expressing in the luminal membrane of the brain capillary endothelial cells, i.e., the bold-brain barrier (BBB). One of the most critical problems in the CNS acting drug discovery and development is that these efflux transport functions generate significantly lower unbound drug concentration in the brain than that of plasma. Therefore, it is a key issue to predict unbound drug concentration gradient between brain and plasma (Kp,uu) across the BBB. Our ultimate goal is to establish a novel strategy to solve this critical problem in the diseased human brain. Recently, we have succeeded to predict Kp,uu for several P-gp substrates in the normal brain of mouse and cynomolgus monkey based on the pharmacoprotemic model analysis combined with the quantitative analysis of P-gp protein expression in the BBB and the in vitro measurement of intrinsic P-gp efflux transport activities (J. Pharmacol. Exp. Ther.,339: 579 (2011), ibid 350: 578 (2014)) together with epileptic model mouse and chronically phenytoin administered normal mouse (Drug Metab Dispos. 42: 1719 (2014)). These results demonstrate that brain distributions of P-gp substrates could be predicted by assuming the in vivo intrinsic P-gp transport activity is the same to that of in vitro. Interestingly, Miller and colleagues have reported that P-gp transport activity is decreased by the treatment of inflammatory cytokines in a short period of time (Mol Pharmacol 69:462, 2006), suggesting that intrinsic P-gp activity is not always constant. Therefore, we have expanded our approach to the analysis of acute phase diseased model, i.e., oxidative stress model. Human brain capillary endothelial cell line (hCMEC/D3), an in vitro human BBB model, was treated with hydrogen peroxide for 20 min. Vinblastine efflux transport activities were significantly reduced. Based on phosphoproteomic analysis of D3 cells, candidate proteins regulating BBB P-gp transport function are identified. Combination of functional analysis and quantitative P-gp protein analysis revealed that caveolin-1 mediated internalization of P-gp protein into cytosolic space is involved in the apparent decrease of BBB P-gp efflux function, suggesting the localization of transporter protein in the brain capillary endothelial cells is also important subject for the deep understanding of disease effect on the BBB transport function. S39 QUANTITATIVE ANALYSIS OF HEPATIC TRANSPORTERS AND DMES DURING GROWTH AND DEVELOPMENT Bhagwat Prasad. University of Washington, Seattle, WA, USA The future of pediatric pharmacology lies on the better prediction of agedependent variability in drug disposition and response to ensure safety and efficacy of drugs in children. In this direction, physiologically based pharmacokinetic and pharmacodynamic models are proving to be promising. However, to establish physiological models, it is important that quantitative abundance data on drug metabolizing enzymes (DMEs) and transporters are obtained from pediatric vs. adult tissues. My presentation will discuss quantitative analysis of hepatic DMEs and transporters during growth and development. We used state-of-the-art quantitative proteomics technique to determine the effect of age on expression of multiple hepatic DMEs and transporters in 136 children and 38 adult subjects. Moreover, interplay of genetic polymorphism and ontogeny will be presented to reflect significance of genotyping studies together with ontogeny studies. An approach established in our laboratory to differentiate biological vs. technical variability, will be presented. These protein quantification data are critical for developing physiologically based pharmacokinetic models to predict drug disposition in children. The
Stefan Oswald. Center of Drug Absorption and Transport (C_DAT), University Medicine of Greifswald, Greifswald, Germany Transporter proteins are nowadays well recognized to affect the pharmacokinetics, efficacy and safety of many drugs in a significant manner. While there is a bunch of data available for hepatic uptake and biliary efflux transporters, the information on intestinal transporters is rather limited. Here, the research activities were mainly focused on ABCB1 (P-glycoprotein), which is known to limit the oral bioavailability of several drugs. However, little is known about the expression and function of other intestinal efflux as well as uptake transporters. The presentation will address the gene and protein expression of clinically relevant transporters along the entire human intestine. Moreover, their pharmacological relevance, the functional interplay of apical and basolateral transporters, their coordinate interaction with metabolizing enzymes as well as their transcriptional regulation will be discussed. S41 ABSTRACT UNAVAILABLE S42 PROTEO-METABOLOMICS IN ACETAMINOPHEN TOXICITY
ELUCIDATING
MECHANISMS
OF
Eric Chun Yong Chan, James Chun Yip Chan. National University of Singapore, Singapore, Singapore The adverse outcomes pathway (AOP) paradigm describes the events beginning from the initial interaction between a toxicant and its cellular target, which activates a cascade of cellular responses, culminating in cellular injury or death. However, the goal of populating the AOP remains elusive due to a lack of mechanistic understanding of the cellular targets and downstream events, which can include activation of signalling cascades, changes in protein post-translational modifications (PTMs), altered gene or protein expression levels, perturbations in organelle function and morphology, etc. In the absence of definitively identified toxicological targets, multi-omics approaches may provide mechanistic insight to pinpoint critical targets and perturbed pathways. The hepatotoxicant acetaminophen (APAP) has long been known to trigger covalent protein binding, yet these do not result in appreciable alterations in protein function that can be causally linked to the toxicity features elicited by APAP. Additionally, although many downstream events such as opening of the mitochondrial permeability transition pore (MPTP) and DNA fragmentation have been identified, the upstream triggers of these events are still unknown. Interestingly, secondary to overt covalent binding, APAP can covertly induce aberrant protein glutathionylation, which is a physiological PTM known to functionally modulate protein activity. To determine if APAP-induced protein glutathionylation is involved in APAP toxicity, we developed and applied the GluICAT proteomic methodology to globally profile aberrant glutathionylation temporally using human HepaRG cells. APAP was found to cause aberrant glutathionylation of critical proteins involved in energy metabolism, protein turnover, defense against cellular stress, calcium dynamics and mitochondrial permeability transition pore (MPTP) opening. A parallel metabolomics investigation permitted integrative proteo-metabolomics and revealed remarkable coherence in the functional deficits elicited by glutathionylation and corresponding metabolic derangements caused by APAP, with particular emphasis on energy metabolism (glycolysis, Krebs cycle, fatty acid b-oxidation and oxidative phosphorylation). Longitudinal analysis indicated these perturbations occurred within 3-6 h and preceded later events such as opening of the mitochondrial permeability transition pore, which suggests a causative, as opposed to an adaptive role. Separately, fibrate pre-treatment and Nacetylcysteine/S-adenosylmethionine supplementation is known to