Formation of CYP3A-specific metabolites of ibrutinib in vitro is correlated with hepatic CYP3A activity and 4ß-hydroxycholesterol/cholesterol ratio.

Ibrutinib is an orally administered Bruton's tyrosine kinase inhibitor approved for the treatment of B-cell malignancies, including chronic lymphocytic leukemia. Ibrutinib is metabolized primarily via oxidation by cytochrome P450 (CYP) 3A4/5 to M37 (the primary active metabolite), M34, and M25. The objectives of this study were to assess the relationship between formation of the major CYP3A-specific ibrutinib metabolites in vitro and hepatic CYP3A activity and protein abundance, and to evaluate the utility of the endogenous CYP3A biomarker, plasma 4ß-hydroxycholesterol (4ß-HC) to cholesterol ratio, to predict ibrutinib metabolite formation in individual cadaveric donors with matching hepatocytes. Ibrutinib (5 µM) was incubated with single-donor human liver microsomes (n = 20) and primary human hepatocytes (n = 15), and metabolites (M37, M34, and M25) were measured by liquid chromatography-tandem mass spectrometry analysis. CYP3A4/5 protein concentrations were measured by quantitative targeted absolute proteomics, and CYP3A activity was measured by midazolam 1'-hydroxylation. Ibrutinib metabolite formation positively correlated with midazolam 1'-hydroxylation in human liver microsomes and hepatocytes. Plasma 4ß-HC and cholesterol concentrations were measured in plasma samples obtained at the time of liver harvest from the same 15 donors with matching hepatocytes. Midazolam 1'-hydroxylation in hepatocytes correlated with plasma 4ß-HC/cholesterol ratio. When an infant donor (1 year old) was excluded based on previous ontogeny studies, M37 and M25 formation correlated with plasma 4ß-HC/cholesterol ratio in the remaining 14 donors (Spearman correlation coefficients [r] 0.62 and 0.67, respectively). Collectively, these data indicate a positive association among formation of CYP3A-specific ibrutinib metabolites in human hepatocytes, hepatic CYP3A activity, and plasma 4ß-HC/cholesterol ratio in the same non-infant donors.

Sinusoidal Organic Anion-Transporting Polypeptide 1B1/1B3 and Bile Canalicular Multidrug Resistance-Associated Protein 2 Play an Essential Role in the Hepatobiliary Disposition of a Synthetic Cyclic Dinucleotide (STING Agonist).

The liver is central to the elimination of many drugs from the body involving multiple processes and understanding of these processes is important to quantitively assess hepatic clearance of drugs. The synthetic STING (STimulator of INterferon Genes protein) agonist is a new class of drugs currently being evaluated in clinical trials as a potential anticancer therapy. In this study, we used ML00960317 (synthetic STING agonist) to investigate the hepatobiliary disposition of this novel molecular entity. A bile-duct cannulated (BDC) rat study indicated that biliary excretion is the major route of elimination for ML00960317 (84% of parent dose in bile). The human biliary clearance using in vitro sandwich cultured human hepatocyte model predicted significant biliary excretion of ML00960317 (biliary excretion index (BEI) of 47%). Moreover, the transport studies using transporter expressing cell lines, hepatocytes, and membrane vesicles indicated that ML00960317 is a robust substrate of OATP1B1, OATP1B3, and MRP2. Using relative expression factor approach, the combined contribution of OATP1B1 (fraction transported (ft) = 0.62) and OATP1B3 (ft = 0.31) was found to be 93% of the active uptake clearance of ML00960317 into the liver. Furthermore, OATP1B1 and OATP1B3-mediated uptake of ML00960317 was inhibited by rifampicin with IC50 of 6.5 and 2.3 µM, respectively indicating an in vivo DDI risk (R value of 1.5 and 2.5 for OATP1B1 and OATP1B3, respectively). These results highlighted an important role of OATP1B1, OATP1B3, and MRP2 in the hepatobiliary disposition of ML00960317. These pathways may act as rate-determining steps in the hepatic clearance of ML00960317 thus presenting clinical DDI risk.

Using Extracellular Vesicles Released by GDNF-Transfected Macrophages for Therapy of Parkinson Disease.

Extracellular vesicles (EVs) are cell-derived nanoparticles that facilitate transport of proteins, lipids, and genetic material, playing important roles in intracellular communication. They have remarkable potential as non-toxic and non-immunogenic nanocarriers for drug delivery to unreachable organs and tissues, in particular, the central nervous system (CNS). Herein, we developed a novel platform based on macrophage-derived EVs to treat Parkinson disease (PD). Specifically, we evaluated the therapeutic potential of EVs secreted by autologous macrophages that were transfected ex vivo to express glial-cell-line-derived neurotrophic factor (GDNF). EV-GDNF were collected from conditioned media of GDNF-transfected macrophages and characterized for GDNF content, size, charge, and expression of EV-specific proteins. The data revealed that, along with the encoded neurotrophic factor, EVs released by pre-transfected macrophages carry GDNF-encoding DNA. Four-month-old transgenic Parkin Q311(X)A mice were treated with EV-GDNF via intranasal administration, and the effect of this therapeutic intervention on locomotor functions was assessed over a year. Significant improvements in mobility, increases in neuronal survival, and decreases in neuroinflammation were found in PD mice treated with EV-GDNF. No offsite toxicity caused by EV-GDNF administration was detected. Overall, an EV-based approach can provide a versatile and potent therapeutic intervention for PD.

A phase I evaluation of the effect of curcumin on dose-limiting toxicity and pharmacokinetics of irinotecan in participants with solid tumors.

Curcumin inhibits UDP-glucuronyltransferases, a primary metabolic pathway for cancer chemotherapeutic agents like irinotecan. Concurrent administration of both agents may exacerbate irinotecan toxicity. We conducted this phase I study to determine the safety of concurrent curcumin and irinotecan administration. Ten participants with advanced solid tumors received one of four doses (1, 2, 3, and 4 g) of a curcumin phosphatidylcholine complex (PC) orally daily, and 200 mg/m2 of i.v. infusion irinotecan on days 1 and 15 of a 28-day cycle, to determine the maximum tolerated dose (MTD) of PC. Thirteen participants received 4 g of PC (MTD) to assess the effect on the pharmacokinetic (PK) properties of irinotecan and its metabolites, SN-38 and SN-38G. Irinotecan, SN-38, and SN-38G exposure equivalence with and without curcumin was assessed using area under the plasma concentration-time curves from 0 to 6 h (AUC0-6h ). Safety assessments and disease responses were also evaluated. The combination of irinotecan and PC was well-tolerated. Because there was no dose limiting toxicity, the maximum dose administered (4 g) was defined as the recommended phase II dose of PC. PC did not significantly alter the plasma exposure and other PK properties of irinotecan and its metabolites. There was no apparent increase in the incidence of irinotecan-associated toxicities. The objective response rate was 3/19 (22%, 95% confidence interval [CI]: 5-39%), median progression free survival and overall survival (n = 23) were 4 months (95% CI: 2.9-8.9 months) and 8.4 months (95% CI: 3.7 - not evaluable [NE]), respectively. Future studies are required to evaluate the efficacy of this combination.

Estradiol influences adenosinergic signaling and nonrapid eye movement sleep need in adult female rats.

Gonadal steroids and gender are risk factors for sleep disruptions and insomnia in women. However, the relationship between ovarian steroids and sleep is poorly understood. In rodent models, estradiol (E2) suppresses sleep in females suggesting that E2 may reduce homeostatic sleep need. The current study investigates whether E2 decreases sleep need and the potential mechanisms that govern E2 suppression of sleep. Our previous findings suggest that the median preoptic nucleus (MnPO) is a key nexus for E2 action on sleep. Using behavioral, neurochemical, and pharmacological approaches, we tested whether (1) E2 influenced the sleep homeostat and (2) E2 influenced adenosine signaling in the MnPO of adult female rats. In both unrestricted baseline sleep and recovery sleep from 6-h sleep deprivation, E2 significantly reduced nonrapid eye movement (NREM) sleep-delta power, NREM-slow wave activity (NREM-SWA, 0.5-4.0 Hz), and NREM-delta energy suggesting that E2 decreases homeostatic sleep need. However, coordinated with E2-induced changes in physiological markers of homeostatic sleep was a marked increase in MnPO extracellular adenosine (a molecular marker of homeostatic sleep need) during unrestricted and recovery sleep in E2-treated but not oil control animals. While these results seemed contradictory, systemically administered E2 blocked the ability of CGS-21680 (adenosine A2A receptor agonist) microinjected into the MnPO to increase NREM sleep suggesting that E2 may block adenosine signaling. Together, these findings provide evidence that E2 may attenuate the local effects of the A2A receptors in the MnPO, which in turn may underlie estrogenic suppression of sleep behavior as well as changes in homeostatic sleep need.

Comparison of Hepatic Transporter Tissue Expression in Rodents and Interspecies Hepatic OCT1 Activity.

Hepatic clearance may be uptake rate limited by organic anion transporting polypeptides (OATPs) and organic cation transporter 1 (OCT1). While comparison of OATP activity has been investigated across species, little has been reported for OCT1. Additionally, while data on interspecies transporter expression in the liver exist, quantitative comparison of these transporters in multiple tissues is lacking. In the current research, the pharmacokinetics of OCT1 substrates (sumatriptan and metformin) were assessed in Oct knockout rats for comparison with previous Oct1/2-/- mice data and OCT1 pharmacogenetics in humans. Effect of OCT1 inhibitors verapamil and erlotinib on OCT1 substrate liver partitioning was also evaluated in rats. Expression of 18 transporters, including Oatps and Octs, in 9 tissues from mice and rats was quantitated using nanoLC/MS-MS, along with uptake transporters in hepatocytes from 5 species. Interspecies differences in OCT1 activity were further evaluated via uptake of OCT1 substrates in hepatocytes with corresponding in vivo liver partitioning in rodents and monkey. In Oct1-/- rats, sumatriptan hepatic clearance and liver partitioning decreased; however, metformin pharmacokinetics were unaffected. OCT1 inhibitor coadministration decreased sumatriptan liver partitioning. In rodents, Oatp expression was highest in the liver, although comparable expression of Oatps in other tissues was determined. Expression of Octs was highest in the kidney, with liver Oct1 expression comparably lower than Oatps. Liver partitioning of OCT1 substrates was lower in rodents than in monkey, in agreement with the highest OCT1 expression and uptake of OCT1 substrates in monkey hepatocytes. Species-dependent OCT1 activity requires consideration when translating preclinical data to the clinic.

Pregnancy-Related Hormones Increase Nifedipine Metabolism in Human Hepatocytes by Inducing CYP3A4 Expression.

Pregnancy-related hormones (PRH) have emerged as key regulators of hepatic cytochrome P450 (CYP) enzyme expression and function. The impact of PRH on protein levels of CYP3A4 and other key CYP enzymes, and the metabolism of nifedipine (a CYP3A4 substrate commonly prescribed during pregnancy), was evaluated in primary human hepatocytes. Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to PRH (estradiol, estriol, estetrol, progesterone, and cortisol), individually or in combination as a cocktail. Absolute protein concentrations of twelve CYP isoforms in SCHH membrane fractions were quantified by nanoLC-MS/MS, and metabolism of nifedipine to dehydronifedipine in SCHH was evaluated. PRH significantly increased CYP3A4 protein concentrations and nifedipine metabolism to dehydronifedipine in a concentration-dependent manner. CYP3A4 mRNA levels in hepatocyte-derived exosomes positively correlated with CYP3A4 protein levels and dehydronifedipine formation in SCHH. PRH also increased CYP2B6, CYP2C8 and CYP2A6 levels. Our findings demonstrate that PRH increase nifedipine metabolism in SCHH by inducing CYP3A4 expression and alter expression of other key CYP proteins in an isoform-specific manner, and suggest that hepatocyte-derived exosomes warrant further investigation as biomarkers of hepatic CYP3A4 metabolism. Together, these results offer mechanistic insight into the increases in nifedipine metabolism and clearance observed in pregnant women.

Endogenous Coproporphyrin I and III are Altered in Multidrug Resistance-Associated Protein 2-Deficient (TR-) Rats.

Recent studies have focused on coproporphyrin (CP)-I and CP-III (CPs) as endogenous biomarkers for organic anion transporting polypeptides (OATPs). Previous data showed that CPs are also substrates of multidrug resistance-associated protein (MRP/Mrp) 2 and 3. This study was designed to examine the impact of loss of Mrp2 function on the routes of excretion of endogenous CPs in wild-type (WT) Wistar compared to Mrp2-deficient TR- rats. To exclude possible confounding effects of rat Oatps, the transport of CPs was investigated in Oatp-overexpressing HeLa cells. Results indicated that CPs are substrates of rodent Oatp1b2, and that CP-III is a substrate of Oatp2b1. Quantitative targeted absolute proteomic (QTAP) analysis revealed no differences in Oatps, but an expected significant increase in Mrp3 protein levels in TR- compared to WT rat livers. CP-I and CP-III concentrations measured by LC-MS/MS were elevated in TR- compared to WT rat liver, while CP-I and CP-III estimated biliary clearance was decreased 75- and 840-fold in TR- compared to WT rats, respectively. CP-III concentrations were decreased 14-fold in the feces of TR- compared to WT rats, but differences in CP-I were not significant. In summary, the disposition of CPs was markedly altered by loss of Mrp2 and increased Mrp3 function as measured in TR- rats.

Characterization of primary mouse hepatocyte spheroids as a model system to support investigations of drug-induced liver injury.

Primary mouse hepatocytes isolated from genetically defined and/or diverse lines and disease models are a valuable resource for studying the impact of genetic and environmental factors on drug response and disease. However, standard monolayer cultures result in a rapid decline in mouse hepatocyte viability and functionality. Therefore, we evaluated 3D spheroid methodology for long-term culture of primary mouse hepatocytes, initially to support investigations of drug-induced liver injury (DILI). Primary hepatocytes isolated from male and female C57BL/6J mice were used to generate spheroids by spontaneous self-aggregation in ultra-low attachment plates. Spheroids with well-defined perimeters were observed within 5 days after seeding and retained morphology, ATP, and albumin levels for an additional 2 weeks in culture. Global microarray profiling and quantitative targeted proteomics assessing 10 important drug metabolizing enzymes and transporters demonstrated maintenance of mRNA and protein levels in spheroids over time. Activities for 5 major P450 enzymes were also stable and comparable to activities previously reported for human hepatocyte spheroids. Time- and concentration-dependent decreases in ATP and albumin were observed in response to the DILI-causing drugs acetaminophen, fialuridine, AMG-009, and tolvaptan. Collectively, our results demonstrate successful long-term culture of mouse hepatocytes as spheroids and their utility to support investigations of DILI.

Antiretroviral Penetration and Drug Transporter Concentrations in the Spleens of Three Preclinical Animal Models and Humans.

Adequate antiretroviral (ARV) concentrations in lymphoid tissues are critical for optimal antiretroviral therapy (ART). While the spleen contains 25% of the body's lymphocytes, there are minimal data on ARV penetration in this organ. This study quantified total and protein-unbound splenic ARV concentrations and determined whether drug transporters, sex, or infection status were modifiers of these concentrations in animal models and humans. Two humanized mice models (hu-HSC-Rag [n = 36; 18 HIV-positive (HIV+) and 18 HIV-negative (HIV-)] and bone marrow-liver-thymus [n = 13; 7 HIV+ and 6 HIV-]) and one nonhuman primate (NHP) model (rhesus macaque [n = 18; 10 SHIV+ and 8 SHIV-]) were dosed to steady state with ARV combinations. HIV+ human spleens (n = 14) from the National NeuroAIDS Tissue Consortium were analyzed postmortem (up to 24 h postdose). ARV concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), drug transporter concentrations were measured with LC-MS proteomics, and protein binding in NHP spleens was determined by rapid equilibrium dialysis. Mice generally had the lowest splenic concentrations of the three species. Protein binding in splenic tissue was 6 to 96%, compared to 76 to 99% in blood plasma. NHPs had quantifiable Mrp4, Bcrp, and Ent1 concentrations, and humans had quantifiable ENT1 concentrations. None significantly correlated with tissue ARV concentrations. There was also no observable influence of infection status or sex. With these dosing strategies, NHP splenic penetration most closely resembled that of humans. These data can inform tissue pharmacokinetic scaling to humans to target HIV reservoirs by identifying important species-related differences.

PLX038: a PEGylated prodrug of SN-38 independent of UGT1A1 activity.

PURPOSE: The purpose of this study was to determine the importance of UGT1A1 activity on the metabolism and pharmacokinetics of a releasable PEG ~ SN-38 conjugate, PLX038A. Irinotecan (CPT-11) is converted to the topoisomerase 1 inhibitor SN-38 by first-pass hepatic metabolism and is converted to its glucuronide SN-38G by UGT1A1. With diminished UGT1A1 activity, the high liver exposure to SN-38 can cause increased toxicity of CPT-11. In contrast, releasable PEG ~ SN-38 conjugates-such as PLX038-release SN-38 in the vascular compartment, and only low levels of SN-38 are expected to enter the liver by transport through the OATP1B1 transporter. METHODS: We measured CPT-11 and PLX038A metabolites in plasma and bile, and determined pharmacokinetics of PLX038A in UGT1A-deficient and replete rats. RESULTS: Compared to CPT-11, treatment of rats with PLX038A results in very low levels of biliary SN-38 and SN-38G, a low flux through UGT1A, and a low SN-38G/SN-38 ratio in plasma. Further, the pharmacokinetics of plasma PLX038A and SN-38 in rats deficient in UGT1A is unchanged compared to normal rats. CONCLUSIONS: The disposition of PEGylated SN-38 is independent of UGT1A activity in rats, and PLX038 may find utility in full-dose treatment of patients who are UGT1A1*28 homozygotes or have metastatic disease with coincidental or incidental liver dysfunction.

Antiretroviral Drug Concentrations in Lymph Nodes: A Cross-Species Comparison of the Effect of Drug Transporter Expression, Viral Infection, and Sex in Humanized Mice, Nonhuman Primates, and Humans.

In a "kick and kill" strategy for human immunodeficiency virus (HIV) eradication, protective concentrations of antiretrovirals (ARVs) in the lymph node are important to prevent vulnerable cells from further HIV infection. However, the factors responsible for drug distribution and concentration into these tissues are largely unknown. Although humanized mice and nonhuman primates (NHPs) are crucial to HIV research, ARV tissue pharmacology has not been well characterized across species. This study investigated the influence of drug transporter expression, viral infection, and sex on ARV penetration within lymph nodes of animal models and humans. Six ARVs were dosed for 10 days in humanized mice and NHPs. Plasma and lymph nodes were collected at necropsy, 24 hours after the last dose. Human lymph node tissue and plasma from deceased patients were collected from tissue banks. ARV, active metabolite, and endogenous nucleotide concentrations were measured by liquid chromatography-tandem mass spectrometry, and drug transporter expression was measured using quantitative polymerase chain reaction and quantitative targeted absolute proteomics. In NHPs and humans, lymph node ARV concentrations were greater than or equal to plasma, and tenofovir diphosphate/deoxyadenosine triphosphate concentration ratios achieved efficacy targets in lymph nodes from all three species. There was no effect of infection or sex on ARV concentrations. Low drug transporter expression existed in lymph nodes from all species, and no predictive relationships were found between transporter gene/protein expression and ARV penetration. Overall, common preclinical models of HIV infection were well suited to predict human ARV exposure in lymph nodes, and low transporter expression suggests primarily passive drug distribution in these tissues. SIGNIFICANCE STATEMENT: During human immunodeficiency virus (HIV) eradication strategies, protective concentrations of antiretrovirals (ARVs) in the lymph node prevent vulnerable cells from further HIV infection. However, ARV tissue pharmacology has not been well characterized across preclinical species used for HIV eradication research, and the influence of drug transporters, HIV infection, and sex on ARV distribution and concentration into the lymph node is largely unknown. Here we show that two animal models of HIV infection (humanized mice and nonhuman primates) were well suited to predict human ARV exposure in lymph nodes. Additionally, we found that drug transporter expression was minimal and-along with viral infection and sex-did not affect ARV penetration into lymph nodes from any species.

Molecular transport through primary human small intestinal monolayers by culture on a collagen scaffold with a gradient of chemical cross-linking.

BACKGROUND: The luminal surface of the small intestine is composed of a monolayer of cells overlying a lamina propria comprised of extracellular matrix (ECM) proteins. The ECM provides a porous substrate critical for nutrient exchange and cellular adhesion. The enterocytes within the epithelial monolayer possess proteins such as transporters, carriers, pumps and channels that participate in the movement of drugs, metabolites, ions and amino acids and whose function can be regulated or altered by the properties of the ECM. Here, we characterized expression and function of proteins involved in transport across the human small intestinal epithelium grown on two different culture platforms. One strategy employs a conventional scaffolding method comprised of a thin ECM film overlaying a porous membrane while the other utilizes a thick ECM hydrogel placed on a porous membrane. The thick hydrogel possesses a gradient of chemical cross-linking along its length to provide a softer substrate than that of the ECM film-coated membrane while maintaining mechanical stability. RESULTS: The monolayers on both platforms possessed goblet cells and abundant enterocytes and were impermeable to Lucifer yellow and fluorescein-dextran (70 kD) indicating high barrier integrity. Multiple transporter proteins were present in both primary-cell culture formats at levels similar to those present in freshly isolated crypts/villi; however, expression of breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the monolayers on the conventional scaffold was substantially less than that on the gradient cross-linked scaffold and freshly isolated crypts/villi. Monolayers on the conventional scaffold failed to transport the BCRP substrate prazosin while cells on the gradient cross-linked scaffold successfully transported this drug to better mimic the properties of in vivo small intestine. CONCLUSIONS: The results of this comparison highlight the need to create in vitro intestinal transport platforms whose characteristics mimic the in vivo lamina propria in order to accurately recapitulate epithelial function.

Factors Affecting Interindividual Variability of Hepatic UGT2B17 Protein Expression Examined Using a Novel Specific Monoclonal Antibody.

Accurate quantification of the metabolic enzyme uridine diphospho-glucuronosyltransferase (UGT) UGT2B17 has been hampered by the high sequence identity with other UGT2B enzymes (as high as 94%) and by the lack of a specific antibody. Knowing the significance of the UGT2B17 pathway in drug and hormone metabolism and cancer, we developed a specific monoclonal antibody (EL-2B17mAb), initially validated by the lack of detection in liver microsomes of an individual carrying no UGT2B17 gene copy and in supersomes expressing UGT2B enzymes. Immunohistochemical detection in livers revealed strong labeling of bile ducts and variable labeling of hepatocytes. Expression levels assessed by immunoblotting were highly correlated to mass spectrometry-based quantification (r = 0.93), and three major expression patterns (absent, low, or high) were evidenced. Livers with very low expression were carriers of the functional rs59678213 G variant, located in the binding site for the transcription factor forkhead box A1 (FOXA1) of the UGT2B17 promoter. The highest level of expression was observed for individuals carrying at least one rs59678213 A allele. Multiple regression analysis indicated that the number of gene copies explained only 8% of UGT2B17 protein expression, 49% when adding rs59678213, reaching 54% when including sex. The novel EL-2B17mAb antibody allowed specific UGT2B17 quantification and exposed different patterns of hepatic expression. It further suggests that FOXA1 is a key driver of UGT2B17 expression in the liver. The availability of this molecular tool will help characterize the UGT2B17 level in various disease states and establish more precisely the contribution of the UGT2B17 enzyme to drug and hormone metabolism.

Quantification of membrane transporter proteins in human lung and immortalized cell lines using targeted quantitative proteomic analysis by isotope dilution nanoLC-MS/MS.

Information is needed on the expression of transporters in lung to inform drug development and therapeutic decisions. Much of the information currently available is from semiquantitative gene expression or immunometric densitometry studies reported in the literature. NanoLC-MS/MS (MRM mode) isotope dilution targeted quantitative proteomics was used here to quantify twelve selected transporters in fresh human lung membrane fraction samples and in the membrane fraction of selected immortalized human lung epithelial cell line samples. Fractionation was undertaken by homogenization in crude membrane lysis buffer followed by differential centrifugation of the homogenate. In lung membranes we found OATPs to be the most highly expressed transporters of those measured, followed by PEPT2 and ABCs (P-gp & BCRP). SLC22A transporters (OCTs 2 & 3 and OCTN1) were also found to be expressed. OATP2A1, also known as the prostaglandin transporter, was the most highly expressed transporter, being low in two subjects who were at least occasional smokers. One subject, a non-smoker, had an OATP2A1 concentration that was 8.4 times higher than the next nearest concentration, which itself was higher than the concentration of any other transporter. OATP2A1 is known, from gene expression and animal functional studies, to be present in lung. These results inform the understanding of xenobiotic disposition in the lung and show the distinct profile of transporters in lung compared to other tissues.

Efflux transporter breast cancer resistance protein dominantly expresses on the membrane of red blood cells, hinders partitioning of its substrates into the cells, and alters drug-drug interaction profiles.

1. Red blood cell (RBC) partitioning is important in determining pharmacokinetic and pharmacodynamic properties of a compound; however, active transport across RBC membranes is not well understood, particularly without transporter-related cell membrane proteomics data. 2. In this study, we quantified breast cancer resistance protein (BCRP/Bcrp) and MDR1/P-glycoprotein (P-gp) protein expression in RBCs from humans, monkeys, dogs, rats and mice using nanoLC/MS/MS, and evaluated their effect on RBC partitioning and plasma exposure of their substrates. BCRP-specific substrate Cpd-1 and MDR1-specific substrate Cpd-2 were characterized using Caco-2 Transwell® system and then administered to Bcrp or P-gp knockout mice. 3. The quantification revealed BCRP/Bcrp but not MDR1/P-gp to be highly expressed on RBC membranes. The knockout mouse study indicated BCRP/Bcrp pumps the substrate out of RBCs, lowering its partitioning and thus preventing binding to intracellular targets. This result was supported by a Cpd-1 and Bcrp inhibitor ML753286 drug-drug interaction (DDI) study in mice. Because of enhanced partitioning of Cpd-1 into RBCs after BCRP/Bcrp inhibition, Cpd-1 plasma concentration changed much less extent with genetic or chemical knockout of Bcrp albeit marked blood concentration increase, suggesting less DDI effect. 4. This finding is fundamentally meaningful to RBC partitioning, pharmacokinetics and DDI studies of BCRP-specific substrates.

Procedural outcomes of fluoroless catheter ablation outside the traditional catheterization lab.

AIMS: Non-fluoroscopic catheter ablation is becoming routine. In experienced centres, fluoroscopy is rarely required. The use of a traditional catheterization lab (cath lab) may no longer be necessary. We began performing catheter ablations at a paediatric centre outside the traditional cardiac cath lab in 2013. The purpose of this study was to compare procedural features of paediatric catheter ablation performed outside the cath lab to those performed within a cath lab. METHODS AND RESULTS: We prospectively looked at patients presenting to the paediatric centre with supraventricular tachycardia (SVT) undergoing catheter ablation outside the cath lab in a standard operating room (OR group). We compared retrospectively to a control group matched for age, type, and location of arrhythmia who had ablations in a traditional cath lab (CL group). Catheter visualization was exclusively by electro-anatomic mapping. Fifty-nine patients with SVT underwent catheter ablation in the OR from October 2013 to December 2015. Thirty-three patients had accessory pathways, 29 were manifest, and 13 of those were left sided. Twenty-six had atrioventricular nodal re-entrant tachycardia. Transseptal puncture with transoesophageal echocardiography guidance was used for 10 left-sided pathways, whereas the other 3 had patent foramen ovales. Procedure time did not differ significantly between groups (OR group mean 131 min, range 57-408; CL group mean 152 min, range 68-376; P = 0.12). Acute success was similar in both groups [OR group: 58/59 (98.3%) and CL group: 57/59 (96.6%)]. There were no major complications in either group. There was no fluoroscopy used in either group. CONCLUSION: Although performing paediatric catheter ablations outside the traditional cath lab is early in our experience, we produced similar outcomes and results without encountering procedural difficulties of performing ablations in a non-conventional setting. Larger multi-centred trials will be essential to determine the feasibility of this practice.

Quantification of Four Efflux Drug Transporters in Liver and Kidney Across Species Using Targeted Quantitative Proteomics by Isotope Dilution NanoLC-MS/MS.

PURPOSE: The expression levels of several efflux drug transporters in the liver and kidney were evaluated across species to address potential roles of the transporters in species dependent excretion of drugs and their metabolites. METHODS: Four efflux transporters, namely MDR1/P-gp, BCRP/Bcrp, MRP2/Mrp2 and MRP3/Mrp3 in liver and kidney in three preclinical species and humans were quantified using targeted quantitative proteomics by isotope dilution nanoLC-MS/MS. RESULTS: In liver, the level of P-gp was highest in monkey and lowest in rat. The concentration of BCRP/Bcrp was highest in dog followed by monkey. MRP2/Mrp2 level was highest in monkey and rat, whereas MRP3/Mrp3 levels were similar in human, monkey and dog. In the kidney, the concentrations of MDR1/P-gp in human and monkey were roughly 2 to 3-fold higher than in rat and dog. In rat, BCRP/Bcrp concentrations were substantially higher than in any of the other species. MRP2/Mrp2 concentrations were similar across species, whereas expression of MRP3/Mrp3 was highest in rat. CONCLUSION: Overall, the results indicated that the pattern of hepatic and renal expression of the transporters was quite species dependent. This information should be helpful in the estimation of transport mediated drug and metabolites excretion in liver and kidney across species.

UGT concentrations in human rectal tissue after multidose, oral curcumin.

In vitro studies have demonstrated that curcumin is a substrate for uridine diphosphate glucuronosyltransferase (UGTs), with a putative ability to both induce expression and inhibit function, highlighting the potential for interaction with some drugs. Therefore, we sought to evaluate the effect of oral curcumin on intestinal UGT expression. Healthy volunteers, ages 40-80 years, who had received recent screening colonoscopy were recruited. Participants did not have any gastrointestinal or bleeding disorders, lab abnormalities, or recent antibiotic use. All participants received daily curcuminoid extract, 4 g, for 30 days. Untreated, rectal mucosal pinch biopsies were obtained at baseline and at 30 days. Microsomes were prepared from biopsy samples, using sequential centrifugation. Quantification of 14 UGT 2As and 2Bs was performed by LC-MS/MS(MS, mass spectrometry), using quantitative- targeted absolute proteomics. Lowest LODs were ~0.1 pmol/mg protein. Comparisons were performed using Wilcoxon signed-rank test. Paired baseline and 30 days biopsy samples were available for 38 participants. UGTs 1A10 and 2B17 were detected in 35 and 33 paired samples, respectively, while all other UGTs were below the limit of quantification (BLOQ). Median baseline UGT1A10 concentration was 0.60 pmol/mg (95% CI:0.32-0.92), and 0.60 pmol/mg (95% CI:0.43-1.00) after 30 days (P = 0.23). For UGT2B17, median baseline concentration was 0.83 pmol/mg (95% CI:0.32-1.62), and 1.18 pmol/mg (95% CI:0.39-1.77) after 30 days (P = 0.24). We found no differences in rectal mucosal UGT concentrations before and after 30 days of oral curcumin administration, indicating that daily curcumin use is unlikely to alter colonic UGT expression. Distal gut biopsies may not accurately reflect the proximal gut environment where UGT expression and curcumin concentrations may be higher.

Quantitative profiling of human renal UDP-glucuronosyltransferases and glucuronidation activity: a comparison of normal and tumoral kidney tissues.

Renal metabolism by UDP-glucuronosyltransferase (UGT) enzymes is central to the clearance of many drugs. However, significant discrepancies about the relative abundance and activity of individual UGT enzymes in the normal kidney prevail among reports, whereas glucuronidation in tumoral kidney has not been examined. In this study, we performed an extensive profiling of glucuronidation metabolism in normal (n = 12) and tumor (n = 14) kidneys using targeted mass spectrometry quantification of human UGTs. We then correlated UGT protein concentrations with mRNA levels assessed by quantitative polymerase chain reaction and with conjugation activity for the major renal UGTs. Beyond the wide interindividual variability in expression levels observed among kidney samples, UGT1A9, UGT2B7, and UGT1A6 are the most abundant renal UGTs in both normal and tumoral tissues based on protein quantification. In normal kidney tissues, only UGT1A9 protein levels correlated with mRNA levels, whereas UGT1A6, UGT1A9, and UGT2B7 quantification correlated significantly with their mRNA levels in tumor kidneys. Data support that posttranscriptional regulation of UGT2B7 and UGT1A6 expression is modulating glucuronidation in the kidney. Importantly, our study reveals a significant decreased glucuronidation capacity of neoplastic kidneys versus normal kidneys that is paralleled by drastically reduced UGT1A9 and UGT2B7 mRNA and protein expression. UGT2B7 activity is the most repressed in tumors relative to normal tissues, with a 96-fold decrease in zidovudine metabolism, whereas propofol and sorafenib glucuronidation is decreased by 7.6- and 5.2-fold, respectively. Findings demonstrate that renal drug metabolism is predominantly mediated by UGT1A9 and UGT2B7 and is greatly reduced in kidney tumors.