Maarten Vliegen

In Vivo Visualization and Quantification of (Disturbed) Oatp-Mediated Hepatic Uptake and Mrp2-Mediated Biliary Excretion of 99mTc-Mebrofenin in Mice

Hepatic transport of 99mTc-mebrofenin through organic anion transport protein 1a and 1b (Oatp1a/1b) and multidrug resistance protein 2 (Mrp2) was investigated by small-animal SPECT. On the basis of the results, a noninvasive method to visualize and quantify disturbances in hepatic transport is proposed. Methods: Friend virus B wild-type mice (untreated, bile duct–ligated, vehicle- or rifampicin-treated) and strain-matched knockout mice unable to express the uptake transporters Oatp1a/1b (Slco1a/1b−/−/−/−) or the efflux transporter Mrp2 (Abcc2−/−) were intravenously injected with 99mTc-mebrofenin (n = 3 per group). After dynamic small-animal SPECT and short CT acquisitions, time–activity curves of the liver and of the gallbladder and intestines were obtained and correlated with direct blood samples. Results: Normal hepatobiliary clearance of 99mTc-mebrofenin was severely impaired in the bile duct–ligated animal, as evidenced by elevated hepatic tracer levels. In Slco1a/1b−/−/−/− mice, a lower area under the curve (AUC) for the liver (P = 0.014) was obtained and no activity was detected in the gallbladder and intestines. Renal rerouting was observed, along with an increase in the blood AUC (P = 0.01). Abcc2−/− mice had a higher liver AUC (P = 0.009), a delayed emergence time of 99mTc-mebrofenin in the gallbladder (P = 0.009), and a lower AUC for the gallbladder and intestines (P = 0.001). The blood curve was similar to that of wild-type mice. 99mTc-mebrofenin disposition was altered after rifampicin treatments. We observed a dose-dependent delayed time point at which tracer maximized in liver, an increased AUC for liver, and a lower AUC for gallbladder and intestines (P = 0.042, 0.034, and 0.001, respectively, highest dose). Emergence in the gallbladder occurred later (P = 0.009, highest dose), and blood AUC was higher (P = 0.006). Conclusion: The current study visualized and quantified hepatic uptake and biliary efflux of 99mTc-mebrofenin. Our results demonstrated the possibility of discriminating, on a quantitative level, between lack of functional activity of sinusoidal uptake versus that of biliary efflux transporters.

A novel radioligand for the ATP-gated ion channel P2X7: [3H] JNJ-54232334.

The ATP-gated ion channel P2X7 has emerged as a potential central nervous system (CNS) drug target based on the hypotheses that pro-inflammatory cytokines such as IL-1β that are released by microglia, may contribute to the etiology of various disorders of the CNS including depression. In this study, we identified two closely related P2X7 antagonists, JNJ-54232334 and JNJ-54140515, and then tritium labeled the former to produce a new radioligand for P2X7. JNJ-54232334 is a high affinity ligand for the rat P2X7 with a pKi of 9.3±0.1. In rat cortical membranes, [3H] JNJ-54232334 reached saturable binding with equilibrium dissociation (Kd) constant of 4.9±1.3 nM. The compound displayed monophasic association and dissociation kinetics with fast on and off rates. In rat brain sections, specific binding of [3H] JNJ-54232334 was markedly improved compared to the previously described P2X7 radioligand, [3H] A-804598. In P2X7 knockout mouse brain sections, [3H] A-804598 bound to non-P2X7 binding sites in contrast to [3H] JNJ-54232334. In rat or wild type mouse brain sections [3H] JNJ-54232334 bound in a more homogenous and region independent manner. The ubiquitous expression of P2X7 receptors was confirmed with immunohistochemistry in rat brain sections. The partial displacement of [3H] A-804598 binding resulted in the underestimation of the level of ex vivo P2X7 occupancy for JNJ-54140515. Higher levels of P2X7 ex vivo occupancy were measured using [3H] JNJ-54232334 due to less non-specific binding. In summary, we describe [3H] JNJ-54232334 as a novel P2X7 radioligand, with improved properties over [3H] A-804598.

Iridium(I)-catalyzed ortho-directed hydrogen isotope exchange in continuous-flow reactors

Deuterated compounds are important in drug discovery and development, e.g., to prepare the so-called stable isotopically labeled internal standards. However, their preparation can be challenging, and its scalability could be difficult with regard to safety aspects. Herein, we report the first continuous-flow iridium(I)-catalyzed ortho-directed hydrogen-deuterium exchange reaction in microreactors. An immobilized iridium(I) catalyst was loaded in continuous- flow microreactors, such as continuous stirred tank reactors and packed-bed reactors. Best results were obtained in a packed-bed reactor allowing deuterium labeling up to M +7.

Synthesis, in vitro and in vivo small-animal SPECT evaluation of novel technetium labeled bile acid analogues to study (altered) hepatic transporter function.

INTRODUCTION Hepatobiliary transport mechanisms are crucial for the excretion of substrate toxic compounds. Drugs can inhibit these transporters, which can lead to drug-drug interactions causing toxicity. Therefore, it is important to assess this early during the development of new drug candidates. The aim of the current study is the (radio)synthesis, in vitro and in vivo evaluation of a technetium labeled chenodeoxycholic and cholic acid analogue: [(99m)Tc]-DTPA-CDCA and [(99m)]Tc-DTPA-CA, respectively, as biomarker for disturbed transporter functionality. METHODS [99mTc]-DTPA-CDCA([(99m)Tc]-3a) and [99mTc]-DTPA-CA ([(99m)Tc]-3b) were synthesized and evaluated in vitro and in vivo. Uptake of both tracers was investigated in NTCP, OCT1, OATP1B1, OATP1B3 transfected cell lines. Km and Vmax values were determined and compared to [(99m)Tc]-mebrofenin ([(99m)Tc]-MEB). Efflux was investigated by means of CTRL, MRP2 and BSEP transfected inside-out vesicles. Metabolite analysis was performed using pooled human liver S9. Wild type (n=3) and rifampicin treated (n=3) mice were intravenously injected with 37MBq of tracer. After dynamic small-animal SPECT and short CT acquisitions, time-activity curves of heart, liver, gallbladder and intestines were obtained. RESULTS We demonstrated that OATP1B1 and OATP1B3 are the involved uptake transporters of both compounds. Both tracers show a higher affinity compared to [(99m)Tc]-MEB, but are in a similar range as endogenous bile acids for OATP1B1 and OATP1B3. [(99m)Tc]-3a shows higher affinities compared to [(99m)Tc]-3b. Vmax values were lower compared to [(99m)Tc]-MEB, but in the same range as endogenous bile acids. MRP2 was identified as efflux transporter. Less than 7% of both radiotracers was metabolized in the liver. In vitro results were confirmed by in vivo results. Uptake in the liver and efflux to gallbladder + intestines and urinary bladder of both tracers was observed. Transport was inhibited by rifampicin. CONCLUSION The involved transporters were identified; both tracers are taken up in the hepatocytes by OATP1B1 andOATP1B3 with Km and Vmax values in the same range as endogenous bile acids and are secreted into bile canaliculi via MRP2. Dynamic small-animal SPECT imaging can be a useful noninvasive method of visualizing and quantifying hepatobiliary transporter functionality and disturbances thereof in vivo, which could predict drug pharmacokinetics.

Quantitative LC-MS/MS analysis of azide and azidoalanine in in vitro samples following derivatisation with dansyl chloride

An LC-MS/MS method for the simultaneous quantification of azide and azidoalanine in bacterial and mammalian cellular systems was developed to support mutagenicity evaluation of azido containing nucleoside drugs. With the aim to enhance ionization efficiency and chromatographic retention, derivatisation with dansyl chloride was optimized; study samples (diluted 1/10) were derivatised for 2 h at room temperature and subsequently analysed with reversed phase liquid chromatography in combination with electrospray mass spectrometry in the positive ion mode. Good linearity and accuracy were obtained with a lower limit of quantification of 10.0 ng mL−1 for both analytes. The method was applied in mutagenicity experiments and showed time-dependent release of azide, but no formation of azidoalanine could be detected.