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Effect of rifampicin on the distribution of [11C]erlotinib to the liver, a translational PET study in humans and in mice martin bauer, Alexander Traxl, Akihiro Matsuda, Rudolf Karch, Cecile Philippe, Lukas Nics, EvaMaria Klebermass, Beatrix Wulkersdorfer, Maria Weber, Stefan Poschner, Nicolas Tournier, Walter Jäger, Wolfgang Wadsak, Marcus Hacker, Thomas Wanek, Markus Zeitlinger, and Oliver Langer Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00588 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 6, 2018

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Molecular Pharmaceutics

Effect of rifampicin on the distribution of [11C]erlotinib to the liver, a translational PET study in humans and in mice Martin Bauer,1 Alexander Traxl,2 Akihiro Matsuda,1 Rudolf Karch,3 Cécile Philippe,4 Lukas Nics,4 Eva-Maria Klebermass,4 Beatrix Wulkersdorfer,1 Maria Weber,1 Stefan Poschner,5 Nicolas Tournier,6 Walter Jäger,5 Wolfgang Wadsak,4,7 Marcus Hacker,4 Thomas Wanek,2 Markus Zeitlinger,1 Oliver Langer1,2,4,*

1

2

Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria Center for Health & Bioresources, AIT Austrian Institute of Technology GmbH,

Seibersdorf, Austria 3

Center for Medical Statistics, Informatics and Intelligent Systems, Medical University of

Vienna, Austria 4

Department of Biomedical Imaging und Image-guided Therapy, Division of Nuclear

Medicine, Medical University of Vienna, Vienna, Austria 5

Department of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna, Austria

6

IMIV, CEA, Inserm, CNRS, Université Paris-Sud, Université Paris Saclay, CEA-SHFJ,

Orsay, France 7

Center for Biomarker Research in Medicine - CBmed GmbH, Graz, Austria

Corresponding author: Oliver Langer, Department of Clinical Pharmacology, Medical University of Vienna, Währinger-Gürtel 18-20, 1090 Vienna, Austria, Tel.: +43(0) 40400-29810, E-mail: [email protected]

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ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract Organic anion-transporting polypeptides (OATPs) mediate the uptake of various drugs from blood into the liver in the basolateral membrane of hepatocytes. Positron emission tomography (PET) is a potentially powerful tool to assess the activity of hepatic OATPs in

vivo, but its utility critically depends on the availability of transporter-selective probe substrates. We have shown before that among the three OATPs expressed in hepatocytes (OATP1B1, OATP1B3 and OATP2B1) [11C]erlotinib is selectively transported by OATP2B1. In contrast to OATP1B1 and OATP1B3, OATP2B1 has not been thoroughly explored yet and no specific probe substrates are currently available. To assess if the prototypical OATP inhibitor rifampicin can inhibit liver uptake of [11C]erlotinib in vivo, we performed [11C]erlotinib PET scans in 6 healthy volunteers without and with intravenous pretreatment with rifampicin (600 mg). In addition, FVB mice underwent [11C]erlotinib PET scans without and with concurrent intravenous infusion of high-dose rifampicin (100 mg/kg). Rifampicin caused a moderate reduction in the liver distribution of [11C]erlotinib in humans, while a more pronounced effect of rifampicin was observed in mice, in which rifampicin plasma concentrations were higher than in humans. In vitro uptake experiments in an OATP2B1-overexpressing cell line indicated that rifampicin inhibited OATP2B1 transport of [11C]erlotinib in a concentration-dependent manner with a half-maximum inhibitory concentration of 72.0 ± 1.4 µM. Our results suggest that rifampicin-inhibitable uptake transporter(s) contributed to the liver distribution of [11C]erlotinib in humans and mice and that [11C]erlotinib PET in combination with rifampicin may be used to measure the activity of this/these uptake transporter(s) in vivo. Furthermore, our data suggest that a standard clinical dose of rifampicin may exert in vivo a moderate inhibitory effect on hepatic OATP2B1.

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Keywords: liver, hepatocyte, OATP2B1, positron emission tomography, [11C]erlotinib, rifampicin

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Introduction The liver is a key organ involved in the metabolism and excretion of xenobiotics. In the hepatobiliary excretion of many drugs, there is an interplay between membrane transporters belonging to the solute carrier (SLC) and adenosine triphosphate-binding cassette (ABC) families and metabolic enzymes in the liver.1 Organic anion-transporting polypeptides (OATPs) belonging to the SLCO gene superfamily, such as OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3), were shown to mediate the uptake of drugs from the blood into the liver in the basolateral membrane of hepatocytes. ABC transporters, such as multidrug resistance-associated protein 2 (MRP2, ABCC2), promote the secretion of drug metabolites into bile at the canalicular hepatocyte membrane. Hepatocyte transporters are of great concern in clinical pharmacology as drug-drug interactions (DDIs), polymorphisms in the transporter-encoding genes or liver disease may lead to variability in transporter activities, which can sometimes cause pronounced changes in drug disposition with a potential impact on drug safety and efficacy.2-5 A powerful tool to obtain mechanistic information on the influence of membrane transporters on drug disposition in vivo is positron emission tomography (PET) imaging.6-9 The effect of unlabeled drugs on the tissue distribution and excretion of

11

C- or

18

F-labeled transporter

probe substrates can be assessed using PET in humans.10 The utility of PET for transporter studies critically depends on the availability of transporter-specific probe substrates, which are ideally recognized by a single transporter of interest and which undergo negligible metabolism over the duration of a PET scan. The discovery of such probe substrates is challenging due to the broadly overlapping substrate specificities of most SLC and ABC transporters. Some PET tracers have already been described to study hepatic OATP transporters, such as [11C]telmisartan,11 15R-[11C]TIC-Me,12 [11C]dehydropravastatin,13 [11C]glyburide

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and [11C]rosuvastatin.15 However, most of these probe substrates are taken

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up from blood into the liver by several different OATP subtypes, so that they are not suitable to measure the activity of individual uptake transporters. Typical OATP substrates are anionic compounds with low passive permeability, such as statin drugs, for which hepatic uptake is in many cases almost entirely dependent on OATP transport activity.2 However, there is also evidence that other drug classes, such as molecularly targeted anticancer drugs, are transported by hepatic OATPs.16 We have previously performed PET experiments with the radiolabeled, epidermal growth factor receptor (EGFR)-targeted tyrosine kinase inhibitor [11C]erlotinib in healthy volunteers.17 Erlotinib predominantly undergoes hepatobiliary excretion,18 and we found pronounced decreases in the liver distribution of [11C]erlotinib when subjects were pretreated before the PET scan with an oral therapeutic dose of erlotinib, as compared with injection of a microdose of [11C]erlotinib alone. In vitro experiments revealed that [11C]erlotinib is transported with high affinity (Michaelis constant Km, 0.324 µM) and low capacity by OATP2B1 (SLCO2B1), but not by OATP1B1 and OATP1B3, which suggested that saturation of OATP2B1-mediated liver uptake of [11C]erlotinib may have occurred in subjects pre-treated with oral erlotinib.17 While these data indicated that OATP2B1-mediated liver uptake of erlotinib at therapeutic doses is negligible, they suggested that [11C]erlotinib may have some potential as an OATP2B1 imaging probe.17 This is of considerable interest as OATP2B1 is, in contrast to OATP1B1 and OATP1B3, an underexplored hepatic uptake transporter and no OATP2B1-specific probe substrates are currently available. Apart from the liver, OATP2B1 is also expressed in many other organs (e.g. intestine, lung, heart and at the blood-brain barrier).19-22 Some studies have assessed OATP2B1-mediated drug-drug interactions (DDIs) and the impact of SLCO2B1 polymorphisms on the disposition of OATP2B1-substrate drugs, but only for the intestine, and no data are available for the liver.23-

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The availability of an OATP2B1-specific PET probe substrate may therefore provide a

better understanding of the role of OATP2B1 in the hepatic clearance of drugs. The aim of the present study was to determine if the prototypical OATP inhibitor rifampicin, which is commonly used for in vivo DDI studies involving OATPs,26 can inhibit hepatic uptake of [11C]erlotinib. To this end, we performed [11C]erlotinib PET scans in healthy volunteers without and with intravenous (i.v.) pre-treatment with rifampicin (600 mg). To complement our clinical PET data, we also performed PET imaging in FVB mice without and with concurrent i.v. infusion of high-dose rifampicin (100 mg/kg). In addition, we assessed the inhibitory effect of rifampicin on cellular uptake of [11C]erlotinib in an OATP2B1overexpressing cell line.

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Experimental Section The clinical study was approved by the Ethics Committee of the Medical University of Vienna and by the competent authority, registered under EUDRACT number 2015-00159318 and conducted according to the current version of the Declaration of Helsinki. Written consent was obtained from all subjects. Volunteers were defined as healthy based on physical examination, routine laboratory testing (see Supporting Information, Table S1) and medical history. All subjects were non-smokers and were required to be medication free for at least 14 days before the PET scans. Animal experiments were approved by the national authorities (Amt der Niederösterreichischen Landesregierung), and all study procedures were performed in accordance with the European Communities Council Directive of September 22, 2010 (2010/63/EU).

Radiotracer synthesis [11C]Erlotinib was synthesized as described elsewhere.27 Molar activity at the time of injection was 95 ± 59 GBq/µmol and 72 ± 35 GBq/µmol for clinical and preclinical PET imaging, respectively, and radiochemical purity was > 98%.

Clinical PET imaging Two female and 4 male healthy volunteers (mean age: 41 ± 15 years, mean weight: 69 ± 7 kg, see Supporting Information, Table S1 for demographic data) underwent two dynamic 90min [11C]erlotinib PET scans of the upper abdominal region on an Advance scanner (General Electric Medical Systems, Milwaukee, WI, USA).17 A microdose of [11C]erlotinib was injected as an i.v. bolus over 20 s (382 ± 22 MBq, corresponding to 7.4 ± 6.9 nmol of unlabeled erlotinib). In parallel to PET imaging, serial blood samples were rapidly drawn from the radial artery for the first 2.5 min (2-3 mL each), followed by samples taken at 3.5

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min (4 mL), 5 min (4 mL), 10 min (4 mL), 20 min (18 mL), 30 min (4 mL), 40 min (18 mL), 60 min (9 mL) and 90 min (9 mL) after radiotracer injection. For both PET scans, subjects were asked to fast for at least 4 h before radiotracer injection. An i.v. dose of 600 mg rifampicin (Rifoldin®, Sanofi-Aventis GmbH, Vienna, Austria) was administered over 30 min before the start of the second PET scan. Scans with rifampicin pre-treatment were always performed after scans without rifampicin pre-treatment and the time interval between the two scans was approximately 7 days. Blood samples (4 mL) were collected at 30, 60, 90 and 120 min after the start of the rifampicin infusion. Blood samples were centrifuged to obtain plasma, which was immediately aliquoted and snap frozen on dry ice before stored at -80°C until analysis of rifampicin concentrations. Storage time of plasma samples until analysis was < 5 months. In 5 out of 6 study participants, urine was collected at the end of both PET scans and radioactivity was measured in a gamma-counter.

Blood and metabolite analysis Aliquots of blood and plasma were measured for radioactivity in a gamma-counter, which was cross-calibrated with the PET camera. Plasma samples collected at 20 and 40 min after radiotracer injection were analyzed for radiolabeled metabolites of [11C]erlotinib using a previously described solid-phase extraction procedure.17,

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Due to the low percentage of

radiolabeled metabolites in plasma, total radioactivity counts in blood were considered for PET data analysis. Plasma protein binding of [11C]erlotinib was determined in 3 subjects by incubating plasma samples obtained before each PET scan with [11C]erlotinib followed by ultrafiltration as described previously.17

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Preclinical PET imaging Female wild-type mice with a FVB genetic background (Charles River, Sulzfeld, Germany) weighing 24.2 ± 1.7 g underwent under isoflurane anesthesia a 60-min dynamic PET scan with [11C]erlotinib (29 ± 4 MBq administered via the tail vein, corresponding to 0.6 ± 0.3 nmol of unlabeled erlotinib) as described in detail elsewhere.29 At 30 min before start of the PET scan, animals received via the tail vein rifampicin (40 mg/kg, n = 5) or vehicle (aqua ad injectabilia containing 1% hydrochloric acid, v/v, n = 5) as an i.v. bolus over 1 min followed by a continuous infusion of rifampicin (40 mg/kg/h) or vehicle until the end of the PET scan. At the end of the PET scan, a terminal blood sample was withdrawn from the retro-orbital vein and animals were sacrificed by cervical dislocation while still under deep anesthesia. Plasma was stored at -80°C until analysis of rifampicin concentrations. Storage time of plasma samples until analysis was approximately 2 months.

Determination of rifampicin concentrations in plasma The concentration of rifampicin in plasma was determined by high-performance liquid chromatography (HPLC) using a Dionex “UltiMate 3000” system (Dionex Corp., Sunnyvale, CA) with ultraviolet (UV) detection at 336 nm. Frozen human and mouse plasma samples were thawed at room temperature. After the addition of 600 µL ice-cold acetonitrile to 150 µL of human plasma, the samples were centrifuged (14,000 x g for 5 min) and 50 µL of the clear supernatants were injected onto the HPLC column. For mouse samples, 60 µL of icecold acetonitrile was added to 10 µL of plasma, the samples were centrifuged (14,000 x g for 5 min) and 60 µL of the clear supernatants were diluted with 60 µL of 10 mM ammonium acetate/acetic acid buffer (pH 5.0); 100 µL of these mixtures were then injected onto the HPLC column. Chromatographic separation was carried out at 40°C on a Hypersil BDS-C18 column (5 µm, 250 x 4.6 mm I.D., Thermo Fisher Scientific, Inc, Waltham, MA), preceded

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by a Hypersil BDS-C18 pre-column (5 µm, 10 x 4.6 mm I.D.). The mobile phase consisted of a continuous linear gradient, mixed from 10 mM aqueous ammonium acetate/acetic acid buffer, pH 5.0 (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 1.0 mL/min. Mobile phase B linearly increased from 20% (0 min) to 47% at 7.5 min, further increased to 90% at 8.5 min and was kept constant until 13 min. The percentage of acetonitrile was then decreased to 20% within 0.5 min to equilibrate the column for 6 min before injection of the next sample. Linear calibration curves were generated by spiking drug-free human or mouse plasma with standard solutions of rifampicin to give a concentration range from 0.05 µg to 100 µg/mL for human and 0.5 to 100 µg/mL for mouse plasma (average correlation coefficients for human and mouse plasma: >0.999). The limit of quantification (LOQ) for rifampicin in human plasma was 0.06 µg/mL, that for mouse plasma 0.05 µg/mL; coefficients of accuracy and precision for this compound in mouse and human plasma were < 8%.

Imaging data analysis For the clinical PET data, regions of interest for liver, combined bile duct and gall bladder and left renal cortex were manually delineated on PET summation images using PMOD 3.6 (PMOD Technologies, Zurich, Switzerland). From the dynamic PET data, time-activity curves (TACs) in units of standardized uptake value (SUV = (radioactivity per g/injected radioactivity) x body weight) were generated. The area under the TACs in the blood (AUCblood), the liver (AUCliver), the combined bile duct and gall bladder (AUCbile duct+gall bladder) and the left renal cortex (AUCkidney) from 0 to 90 min after radiotracer injection was calculated using Prism 7.0 software (GraphPad Software, La Jolla, CA, USA). The ratio of AUCliver to AUCblood was calculated as a parameter of [11C]erlotinib liver distribution. The biliary secretion clearance and renal clearance of radioactivity with respect to the blood

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concentration (CLbile,blood and CLrenal,blood, mL/min/kg) or with respect to the liver or kidney concentration (CLbile,liver and CLrenal,kidney, mL/min/kg) was calculated by dividing the total amount of radioactivity in the bile duct and gall bladder or in the urine (in kBq) by AUCblood or AUCliver or AUCkidney (in kBq/mL x min), respectively.30 A graphical analysis method (integration plot)

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was used to estimate the rate constants for transfer of radioactivity from

blood into the liver (kuptake,liver, mL/min/g liver) and from blood into the kidney (kuptake,kidney, mL/min/g kidney) using data measured from 0.75 min to 4.5 min and from 0.5 min to 1.3 min after radiotracer injection, respectively, as described in detail elsewhere.29 For integration plot analysis, arterial blood counts were interpolated to the midpoints of the individual PET time-frames. In addition, a 2-tissue compartment model (see Supporting Information, Figure S1),

which

has

been

previously

described

for

the

radiolabeled

bile

acid

[11C]cholylsarcosine,31 was fitted to the arterial blood and liver PET data using freely available iFit software (www.liver.dk/ifit.html). The fraction of blood in liver tissue was set to a value of 0.25. The kinetic model uses the measured arterial blood data and liver PET data to estimate the time course of radiotracer in the portal vein

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and to determine a flow

weighted mixed input function considering both blood supply from the portal vein and the hepatic artery. The model provides estimates of the rate constants for transfer of radioactivity between the different compartments (see Supporting Information, Figure S1). On the reconstructed mouse PET images, left ventricle of the heart (image-derived blood curve), liver, gall bladder, duodenum, intestine and left kidney were manually outlined using the medical image data examiner software AMIDE

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and TACs were extracted as described

elsewhere.29 It was assumed that the sum of radioactivity in the gall bladder, the duodenum and the intestine represented the radioactivity in the bile excreted from the liver. Integration plot analysis was performed to estimate kuptake,liver and kuptake,kidney using the image-derived

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blood curve as well as the rate constant for biliary secretion of radioactivity (kbile, 1/min) as described before.29 In addition, CLrenal,blood and CLbile,blood were calculated as described above.

In vitro uptake experiments We performed cellular uptake experiments, as described in detail before,17 to determine the half-maximum inhibitory concentration (IC50) of rifampicin for OATP2B1-mediated uptake of [11C]erlotinib. For these experiments, human epidermoid carcinoma A431 cells stably overexpressing OATP2B1 were used, which had been generated as described elsewhere.34 Briefly, OATP2B1-overexpressing cells and A431 cells transfected with the empty vector were pre-incubated for 30 min with vehicle solution (1% DMSO) or different rifampicin concentrations (5, 7, 10, 20, 50, 70, 100 and 200 µM). The cells were washed and then incubated for 7.5 min with [11C]erlotinib (~1 MBq, 0.054 ± 0.004 µM) in presence of vehicle solution or the same rifampicin concentrations as used for pre-incubation. Afterwards, cells were washed and radioactivity retained in the cells was measured in a gamma-counter. Experiments were performed 2 times with 3 technical replicates each. Radioactivity uptake in both cell lines was corrected for radioactive decay and expressed as percent of applied dose per 106 cells. OATP2B1-specific uptake of [11C]erlotinib was determined by subtracting the uptake in cells transfected with the empty vector from the total uptake in cells transfected with OATP2B1. OATP2B1-specific uptake of [11C]erlotinib at different rifampicin concentrations was expressed as percentage of OATP2B1-specific uptake of [11C]erlotinib in presence of vehicle solution and data were fitted with Prism 7.0 software to a sigmoid inhibitory effect model (log(inhibitor) vs. normalized response) assuming a fixed Hill-slope of -1.0.

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Statistical analysis Our study was exploratory, so no sample size calculation was performed. All values are given as mean ± standard deviation (SD). Statistical testing was performed using Prism 7.0 software. Differences in outcome parameters between scans were tested using a 2-sided paired or unpaired t-test. To assess correlations, the Pearson correlation coefficient r was calculated. The level of statistical significance was set to a P value of less than 0.05.

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Results Healthy volunteers underwent two PET scans with a microdose of [11C]erlotinib on two separate study days. The first scan was a baseline scan and the second scan was performed following a 30-min i.v. rifampicin infusion. The rifampicin infusion was well tolerated by all subjects without occurrence of adverse drug reactions. Rifampicin infusion had no influence on the percentage of unchanged [11C]erlotinib in plasma at both studied time points. The percentage of unchanged [11C]erlotinib in plasma was 98.0 ± 0.5% in scan 1 and 98.0 ± 0.9% in scan 2 at 20 min after radiotracer injection and 96.4 ± 1.3% in scan 1 and 96.8 ± 1.3% in scan 2 at 40 min after radiotracer injection. The percentage of plasma protein binding of [11C]erlotinib was not significantly different between the two scans (scan 1: 96.5 ± 1.1%, scan 2: 95.7 ± 1.5%, n = 3). Rifampicin concentrations in plasma were measured in all study subjects except for the first subject (Figure 1). Rifampicin concentrations were highest at the start of the PET scan (corresponding to the end of the rifampicin infusion), ranging from 26.0 to 42.7 µM. During the first 30 min after the end of the rifampicin infusion there was a rapid decline in rifampicin concentrations, followed by a slower elimination during the remainder of the PET scan (Figure 1). At the end of the PET scan, mean rifampicin plasma concentration was 10.8 ± 3.4 µM. In Figure 2, serial PET images of the abdominal region are shown for one subject, indicating reduced radioactivity uptake in the liver at early imaging time points of scan 2 as compared with scan 1 (< 10 min after radiotracer injection). In Figure 3, mean TACs in blood, liver and in the bile duct and gall bladder are shown. In Table 1, pharmacokinetic parameters of [11C]erlotinib are summarized. Blood radioactivity concentrations (AUCblood) were not significantly different between the two scans. AUCliver was significantly lower in scan 2,

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while AUCbile duct+gall bladder was not significantly different between the two scans. Liver-toblood concentration ratios (Kb,liver) over time are shown in Figure 3D. The AUC of the Kb,liver

versus time curve was significantly lower in scan 2. In Figures S2 and S3 of the Supporting Information, liver TACs and Kb,liver values are shown for individual subjects, indicating variability in the effect of rifampicin among different subjects. We determined different parameters to describe [11C]erlotinib distribution from blood into the liver (Table 1). AUCliver/AUCblood ratios were significantly lower in scan 2 (range: -5% to 42%) (Figure 4A). There was a significant positive correlation between the percentage change in AUCliver/AUCblood and unbound rifampicin plasma concentration at the start of the PET scan (Figure 5). We used integration plot analysis to determine kuptake,liver values (see Supporting Information, Figure S4). One subject (p40) displayed slower liver uptake kinetics as compared with the other subjects (see Supporting Information, Figure S2), so that a longer linear uptake phase was considered in the integration plot analysis. Kuptake,liver values were significantly decreased in scan 2 (range: -5% to -34%, Figure 4B). In addition, we used a kinetic model to estimate the rate constant for transfer of radioactivity from blood into liver (K1) (see Supporting Information, Figure S1). The kinetic model provided good fits of the liver PET data (see Supporting Information, Figure S5). K1 values estimated with the kinetic model were approximately two times higher than kuptake,liver values (Table 1) and were in a similar range as the liver blood flow rate in humans (0.81 mL/min/g liver).35 K1 values showed a significant positive correlation with kuptake,liver values (r = 0.630, P = 0.028, slope: 0.291 ± 0.113). Like kuptake,liver, K1 was significantly decreased in scan 2 ranging from -7% to -32% (Figure 4C). There was no significant correlation between the percentage change in K1 or kuptake,liver in scan 2 and unbound rifampicin plasma concentration at the start of the PET scan (not shown). We also estimated the rate constants for backflux of radioactivity from liver into blood (k2) and for biliary secretion of radioactivity (k3) (see Supporting

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Information, Figure S1). While k2 was not different between the two scans, k3 was decreased in 5 out of 6 subjects in scan 2 (range: -11% to -86%), without reaching statistical significance (Table 1). In contrast to the liver, the radioactivity concentration in the kidneys was very low and not significantly different between the two scans (see Supporting Information, Figure S6).

Kuptake,kidney values were not significantly different between scan 1 and scan 2 (Table 1). However, the amount of radioactivity excreted into the urine over the time course of the PET scan was significantly higher in scan 2 than in scan 1 (percentage of the injected dose in urine, scan 1: 0.6 ± 0.2%, scan 2: 1.7 ± 0.2%). Moreover, CLrenal,blood and CLrenal,kidney were significantly higher in scan 2. CLbile,blood showed a trend towards a decrease in scan 2 without reaching statistical significance, while CLbile,liver was unchanged (Table 1). We also studied [11C]erlotinib liver and kidney distribution in FVB mice (n = 5) without and with concurrent i.v. infusion of high-dose rifampicin (100 mg/kg). In Figure S7 of the Supporting Information, mean TACs in blood, liver and intestine and Kb,liver values over time are shown. In Table 2, pharmacokinetic parameters of [11C]erlotinib in mice are summarized. In contrast to humans, AUC values in blood, liver and intestine were significantly higher in the rifampicin- as compared with the vehicle-treated group. However, like in humans, rifampicin treatment significantly reduced AUCliver/AUCblood and kuptake,liver (Table 2). In addition, kbile, CLbile,blood, kuptake,kidney and CLrenal,blood were significantly lower in rifampicintreated mice (Table 2). Mean rifampicin concentration in mouse plasma at the end of the PET scan was 38.6 ± 13.0 µM (n = 3), which was 3.6-fold higher than in humans. We performed in vitro uptake experiments to determine the IC50 of rifampicin for inhibition of OATP2B1-specific transport of [11C]erlotinib in OATP2B1-overexpressing cells. Rifampicin concentration-dependently inhibited OATP2B1-transport of [11C]erlotinib with an estimated IC50 of 72.0 ± 1.4 µM (Figure 6). At rifampicin concentrations of 5 µM and 10 µM,

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which corresponded approximately to the free rifampicin plasma concentration range achieved in vivo, the OATP2B1-specific cellular uptake of [11C]erlotinib was 72 ± 18% and 71 ± 2% of control, respectively.

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Discussion Our previous in vitro data in transporter transfected A431 cells had indicated that [11C]erlotinib is at low concentrations transported by OATP2B1, while no transport by OATP1B1 and OATP1B3 was evident.17 In addition, other studies have shown that erlotinib is not a substrate of organic cation transporter 1 (OCT1, SLC22A1) and organic anion transporter 2 (OAT2, SLC22A7), two other uptake transporters expressed in hepatocytes.36 However, the contribution of OATP2B1 to the cellular uptake of [11C]erlotinib in the OATP2B1 transfected cell line was quite small (~25%), which indicated that cellular uptake of [11C]erlotinib was not only dependent on OATP2B1 but also mediated by passive diffusion.17 On the other hand, in vivo a marked 4-fold reduction in the liver distribution of [11C]erlotinib (AUCliver/AUCblood) was observed in subjects pre-treated with a therapeutic dose of unlabeled erlotinib, presumably due to saturation of OATP2B1 transport activity.17 To further investigate a possible role of OATP2B1 in the hepatic uptake of [11C]erlotinib we sought in the present study to assess the influence of a prototypical OATP inhibitor on liver distribution of [11C]erlotinib. Some drugs have been identified as OATP2B1 inhibitors, such as cyclosporine A, rifampicin, atorvastatin and gemfibrozil, as well as fruit juices.21,

37-39

Rifampicin effectively inhibits hepatic OATP1B1 and OATP1B3 at standard clinical doses, which can be safely administered to healthy volunteers, and is therefore commonly used as a prototypical OATP inhibitor for in vivo clinical DDI studies.40-42 Although some studies have shown that rifampicin can inhibit in vitro OATP2B1-mediated transport of various substrates,38, 43, 44 it is currently not known if rifampicin can inhibit hepatic OATP2B1 in vivo at the plasma concentrations attained after administration of a clinically used dose. Reported

in vitro IC50 values of rifampicin were 90 µM and 80.5 µM for bromosulfophthalein 43, 44 and 65 µM for estrone 3-sulfate

38

as substrates. We found that rifampicin concentration-

dependently inhibited uptake of [11C]erlotinib in an OATP2B1-overexpressing cell line with

19



an IC50 value of 72.0 ± 1.4 µM (Figure 6), which was comparable to the IC50 values published in the literature for other OATP2B1 substrates. The maximum blood concentrations of [11C]erlotinib achieved in our study were in the range of 5 nM, which was well below the previously determined Km value for erlotinib transport by OATP2B1 (0.324 µM),17 which indicated that no saturation of OATP2B1 activity had occurred in our study with the administration of the PET microdose of [11C]erlotinib. We found that [11C]erlotinib displayed good metabolic stability over the time course of the PET scan and that single dose i.v. rifampicin infusion had no influence on the percentage of radiolabeled metabolites of [11C]erlotinib in plasma. This is important because an effect of rifampicin, which is a potent CYP3A4 inducer when dosed repeatedly,40 on erlotinib metabolism may have confounded the interpretation of its OATP-inhibitory effect. Our PET data revealed a significant 22% reduction in the liver distribution of radioactivity (AUCliver/AUCblood) in PET scans performed after rifampicin treatment (Figure 4A, Table 1). The effect of rifampicin was variable, with AUCliver/AUCblood reductions ranging from -5% in subject 37 to -42% in subject 38. Rifampicin plasma concentrations at the start of the PET scan ranged from 26.0 to 42.7 µM (Figure 1). It should be noted that a previous study has shown that rifampicin degrades in plasma when stored at -20°C.45 We stored our plasma samples at -80°C, but it still cannot be excluded that some rifampicin degradation had occurred so that true rifampicin plasma concentrations were higher than the measured ones. When considering an unbound fraction of rifampicin in plasma of 0.2,46 free rifampicin concentrations in plasma ranged from 5.2 to 8.5 µM. This is below the in vitro IC50 value of rifampicin to inhibit OATP2B1 transport of [11C]erlotinib (72.0 µM) (Figure 6).17 Based on the in vitro inhibition curve, predicted percentage reductions in OATP2B1-specific cellular uptake of [11C]erlotinib ranged from -7% to -11% for the free rifampicin plasma concentration range obtained in vivo. This was somewhat lower than the observed percentage

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reduction in AUCliver/AUCblood in scan 2 (-22%). However, for an accurate in vitro-in vivo extrapolation of the OATP2B1-inhibitory effect of rifampicin the abundance of OATP2B1 in the employed transporter-overexpressing cell line needs to be considered,47,

48

which is

currently not known. Importantly, we observed a significant positive correlation between the percentage reduction in AUCliver/AUCblood in scan 2 and unbound rifampicin plasma concentration at the start of the PET scan (Figure 5), which supported the assumption that rifampicin partially inhibited OATP2B1-mediated hepatic uptake of [11C]erlotinib. To further elucidate the influence of rifampicin on liver distribution of [11C]erlotinib, we determined the rate constant for transfer of radioactivity from blood into the liver either with integration plot analysis (kuptake,liver), a commonly used approach for PET data analysis 11-13, 30, or with kinetic modeling (K1). Kuptake,liver values were lower than K1 values. This is because the liver receives dual blood supply via the hepatic artery (~25%) and the portal vein (~75%) and kuptake,liver estimates were based on arterial blood, which overestimated the true blood input function, while the kinetic model considered a flow weighted mixed blood input function.31 Both parameters correlated with each other and were moderately but significantly reduced in scan 2 (Table 1, Figure 4). Initial liver uptake of [11C]erlotinib may only be partly mediated by OATP2B1 and may also occur via passive diffusion, which may explain the lack of a significant correlation between the percentage change in kuptake,liver or K1 in scan 2 and rifampicin plasma concentrations. It is noteworthy that in our previous study, in which oral erlotinib was administered before the PET scan, no effect of erlotinib on K1 of [11C]erlotinib was observed, while the backflux of radioactivity from liver into blood (k2) was increased.17 Depending on the employed inhibitor, uptake transporter inhibition may thus either lead to a decrease in K1 or an increase in k2, which will both result in lower liver concentrations of [11C]erlotinib.

21



The practical utility of a probe substrate for transporter imaging depends on the magnitude of the difference in imaging signal between conditions when the transporter is functional and fully inhibited. As compared with other OATP substrates used for PET, such as [11C]dehydropravastatin 13 and [11C]rosuvastatin,15 the effect of rifampicin administration on [11C]erlotinib liver exposure was moderate. This may be related to the fact that rifampicin is a considerably less potent inhibitor of OATP2B1 than of OATP1B1 and OATP1B3.38 Therefore higher rifampicin dosages may be needed to reveal the overall contribution of OATP2B1 to the hepatic distribution of [11C]erlotinib. To gain an understanding of the effect of higher rifampicin doses on liver distribution of [11C]erlotinib, we performed PET experiments in mice without and with concurrent infusion of high-dose rifampicin (100 mg/kg). These experiments showed that kuptake,liver was reduced by -62% and AUCliver/AUCblood by -41% during rifampicin infusion (Table 2). Although there are species differences between mice and humans in the expression of SLCO transporters in hepatocytes,49 the more pronounced effect of rifampicin at high doses in mice supports the notion that rifampicin-inhibitable uptake transporter(s), such as mouse Oatp1b2,50 Oatp2b1, Oatp1a1 or Oatp1a4, contributed to a significant extent to the liver distribution of [11C]erlotinib. Oatp1a1 and Oatp1a4 are the mouse orthologues of human OATP1A2 (SLCO1A2), which is in humans, however, not expressed in the sinusoidal membrane of hepatocytes, but in epithelial cells of the bile duct. Our preliminary, unpublished data indicate that [11C]erlotinib is not transported by OATP1A2, suggesting that it may also not be a substrate of Oatp1a1 and Oatp1a4. However, further studies in transporter knockout or knockdown mice are needed to elucidate which OATP subtypes mediate hepatic uptake of [11C]erlotinib in mice. In our previous clinical study, in which oral erlotinib was administered before the PET scan, much greater reductions in AUCliver/AUCblood of [11C]erlotinib (~4-fold) were observed than in the present study,17 presumably because erlotinib is a more potent

22


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inhibitor of OATP2B1 than rifampicin.38 If this 4-fold reduction in AUCliver/AUCblood corresponds to the overall contribution of OATP2B1 to the liver uptake of [11C]erlotinib in humans, this probe substrate would possess an acceptable dynamic range for hepatic OATP2B1 imaging, although its sensitivity is most likely lower than that of prototypical anionic (non-subtype selective) OATP substrates with low passive permeability. The putatively limited sensitivity of [11C]erlotinib for OATP2B1 imaging may be outweighed by its remarkable selectivity for OATP2B1 over OATP1B1 and OATP1B3.17 It cannot be excluded, though, that other uptake transporters than OATP2B1 contributed to the liver uptake of [11C]erlotinib. However, even if other uptake transporters were involved, measurement of the rifampicin-inhibitable component of [11C]erlotinib liver distribution may allow for specific assessment of OATP2B1 transport activity in the human liver, provided that potential other uptake transporters are insensitive to inhibition by rifampicin. Erlotinib is a substrate of the canalicular hepatocyte ABC transporters breast cancer resistance protein (BCRP, ABCG2) and P-glycoprotein (P-gp, ABCB1), but not of multidrug resistance-associated protein 2 (MRP2, ABCC2).51, 52 Previous experiments in mice indicated that [11C]erlotinib and possibly its radiolabeled metabolites are secreted from the liver into bile via BCRP.29, 53 In scan 2, we found in 5 out of 6 subjects a reduction in the rate constant for biliary secretion of radioactivity (k3), which was consistent with a BCRP inhibitory effect of rifampicin.54 In line with the human data, high-dose rifampicin infusion led in mice to a 52% reduction in kbile (Table 2). A similar observation has been made by He et al., who found that rifampicin inhibited Bcrp- and Mrp2-mediated biliary secretion of [11C]rosuvastatin in rats.15 Surprisingly, in humans renal clearance of radioactivity (CLrenal,blood and CLrenal,kidney) was approximately 3 times higher after rifampicin infusion, which pointed to a shift from hepatobiliary to renal clearance as a compensatory mechanism for slightly impaired

23



hepatobiliary clearance. This may explain why hepatic uptake transporter inhibition did not lead to an increase in blood radioactivity concentrations in humans (Figure 3A). In contrast to humans, rifampicin administration led in mice to an approximately 4-fold increase in blood radioactivity concentrations (see Supporting Information, Figure S7A) and a pronounced decrease in CLbile,blood and CLrenal,blood (Table 2). Renal clearance of [11C]erlotinib was most likely mediated by other, unknown transport proteins, which were probably not inhibited at the rifampicin plasma concentrations achieved in humans, while they were probably inhibited at the higher rifampicin plasma concentrations achieved in mice. The exact mechanism behind the increase in CLrenal,blood in humans after rifampicin administration is not known, but may point to a short-term upregulation of the function of the transporter(s) involved in renal excretion of [11C]erlotinib in response to rifampicin administration. This observation is remarkable as it supports the concept of an inter-organ regulatory network of SLC and ABC transporter activity to maintain homeostasis in the body.55 To our knowledge, these regulatory mechanisms have so far not been thoroughly investigated and should be subject of future investigations. In conclusion, we used PET in combination with different pharmacokinetic analysis approaches to quantitatively assess the effect of the prototypical OATP inhibitor rifampicin on the liver distribution of [11C]erlotinib in humans and mice. Rifampicin caused a moderate reduction in the liver uptake of [11C]erlotinib in humans, while a more pronounced effect was observed in mice, in which rifampicin plasma concentrations were higher than in humans. In

vitro transport experiments revealed that rifampicin concentration dependently inhibited transport of [11C]erlotinib by human OATP2B1. Our data indicate that rifampicin-inhibitable uptake transporter(s) contributed to the liver distribution of [11C]erlotinib in humans and mice. [11C]Erlotinib PET in combination with rifampicin may be used to assess the activity of this/these uptake transporter(s) in vivo. Furthermore, our data suggest that a standard clinical

24


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dose of rifampicin may exert a moderate inhibitory effect on hepatic OATP2B1. However, further data are needed (e.g. experiments involving knockdown of SLCO2B1/Slco2b1 by siRNA in primary human hepatocytes and in mice) to provide definitive proof that liver uptake of [11C]erlotinib is primarily mediated by OATP2B1.

Acknowledgments This work was supported by the Austrian Science Fund (FWF) [grants KLI 480-B30 and F 3513-B20] and by the Lower Austria Corporation for Research and Education (NFB) [grant LS15-003]. The authors thank Johann Stanek (Department of Clinical Pharmacology) for technical assistance as well as Harald Ibeschitz and the other staff members of the PET center at the Division of Nuclear Medicine for supporting this study. Csilla Özvegy-Laczka and Gergely Szakács (Hungarian Academy of Sciences, Budapest, Hungary) are acknowledged for providing the OATP2B1-overexpressing cell line. The authors are grateful to Peter Marhofer and his team (Department of Anesthesia and Intensive Care Medicine) for their support with arterial cannulation of study participants. The PET imaging group at the Austrian Institute of Technology is acknowledged for supporting this study.

Supporting Information Kinetic model used for analysis of [11C]erlotinib liver PET data. Liver TACs in individual subjects. Kb,liver values over time in individual subjects. Integration plots in individual subjects. Exemplary fits of liver PET data. TACs in the left kidney cortex. TACs in arterial blood, liver and intestine and Kb,liver values over time in mice. Table with demographic data and blood chemistry. This material is available free of charge via the Internet at http://pubs.acs.org.

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Hepatology 2002, 36, (1), 164-72. 47. Ishida, K.; Ullah, M.; Toth, B.; Juhasz, V.; Unadkat, J. D. Successful Prediction of In Vivo Hepatobiliary Clearances and Hepatic Concentrations of Rosuvastatin Using Sandwich-Cultured

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Metab. Dispos. 2018, Jun 11. pii: dmd.118.080770. doi: 10.1124/dmd.118.080770. [Epub ahead of print].

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49. Durmus, S.; van Hoppe, S.; Schinkel, A. H. The impact of Organic Anion-Transporting Polypeptides (OATPs) on disposition and toxicity of antitumor drugs: Insights from knockout and humanized mice. Drug Resist. Updat. 2016, 27, 72-88. 50. Bins, S.; van Doorn, L.; Phelps, M. A.; Gibson, A. A.; Hu, S.; Li, L.; Vasilyeva, A.; Du, G.; Hamberg, P.; Eskens, F.; de Bruijn, P.; Sparreboom, A.; Mathijssen, R.; Baker, S. D. Influence of OATP1B1 function on the disposition of sorafenib-beta-D-glucuronide.

Clin. Transl. Sci. 2017, 10, (4), 271-279. 51. Kodaira, H.; Kusuhara, H.; Ushiki, J.; Fuse, E.; Sugiyama, Y. Kinetic analysis of the cooperation of P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp/Abcg2) in limiting the brain and testis penetration of erlotinib, flavopiridol, and mitoxantrone. J. Pharmacol. Exp. Ther. 2010, 333, (3), 788-96. 52. Marchetti, S.; de Vries, N. A.; Buckle, T.; Bolijn, M. J.; van Eijndhoven, M. A.; Beijnen, J. H.; Mazzanti, R.; van Tellingen, O.; Schellens, J. H. Effect of the ATP-binding cassette drug transporters ABCB1, ABCG2, and ABCC2 on erlotinib hydrochloride (Tarceva) disposition in in vitro and in vivo pharmacokinetic studies employing Bcrp1-//Mdr1a/1b-/- (triple-knockout) and wild-type mice. Mol. Cancer Ther. 2008, 7, (8), 22807. 53. Traxl, A.; Wanek, T.; Mairinger, S.; Stanek, J.; Filip, T.; Sauberer, M.; Müller, M.; Kuntner, C.; Langer, O. Breast cancer resistance protein and P-glycoprotein influence in vivo disposition of 11C-erlotinib. J. Nucl. Med. 2015, 56, (12), 1930-1936. 54. Te Brake, L. H.; Russel, F. G.; van den Heuvel, J. J.; de Knegt, G. J.; de Steenwinkel, J. E.; Burger, D. M.; Aarnoutse, R. E.; Koenderink, J. B. Inhibitory potential of tuberculosis drugs on ATP-binding cassette drug transporters. Tuberculosis 2016, 96, 150-7.

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55. Nigam, S. K. What do drug transporters really do? Nat. Rev. Drug Discov. 2015, 14, (1), 29-44.

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Figure Legends Figure 1. Concentration-time curves of rifampicin in arterial plasma of the individual study participants. Rifampicin (600 mg) was administered as an i.v. infusion over 30 min and PET scanning was performed from 30 min to 120 min after the start of the rifampicin infusion. In one study participant (subject 37), rifampicin plasma concentrations were not determined.

Figure 2. Serial PET images of the abdominal region at different time points after [11C]erlotinib injection in one study participant (subject 38) for the baseline scan (scan 1) and the scan recorded after i.v. rifampicin infusion (scan 2). Radioactivity concentration is expressed as standardized uptake value (SUV) and radiation scale is set from 0 to 30. Anatomical structures are labeled with arrows (L, liver; GB, gall bladder; C, colon).

Figure 3. Mean time-activity curves (standardized uptake value (SUV) ± SD, n = 6) in arterial blood (A), liver (B), and bile duct and gall bladder (C) for the baseline scan (scan 1) and the scan recorded after i.v. rifampicin infusion (scan 2). In D, the mean liver-to-blood concentration ratio (Kb,liver) over time is shown for both scans. Figure 4. Outcome parameters for [11C]erlotinib liver distribution in individual study participants for the baseline scan (scan 1) and the scan recorded after i.v. rifampicin infusion (scan 2). In A, the liver-to-blood AUC ratio of radioactivity (AUCliver/AUCblood) is shown. The rate constant for transfer of radioactivity from the blood into the liver determined either with integration plot analysis (kuptake,liver, mL/min/g liver) or kinetic modeling (K1, mL/min/g liver) is shown in B and C, respectively.*, P < 0.05, ** P < 0.01, 2-sided paired t-test.

Figure 5. Correlation of the percentage change in AUCliver/AUCblood in scan 2 with unbound rifampicin plasma concentration (µM) at the start of the PET scan (r = Pearson correlation coefficient). An unbound rifampicin fraction in plasma of 0.2 was assumed.46

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Figure 6. Concentration-dependent inhibition of [11C]erlotinib uptake in OATP2B1overexpressing A431 cells. Uptake of [11C]erlotinib was measured at 37 °C for 7.5 min in OATP2B1-overexpressing A431 cells and in A431 cells transfected with the empty vector in the absence or presence of different concentrations of rifampicin. OATP2B1-specific uptake of [11C]erlotinib was obtained by subtracting uptake in cells transfected with the empty vector from total uptake in OATP2B1-transfected cells. The shown data are taken from two independent experiments and each point represents one individual experiment. Broken line represents best fit to the data according to a sigmoid inhibitory effect model. Vertical dotted line indicates the estimated IC50 value, which is stated with its standard error.

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Table 1. Pharmacokinetic parameters of [11C]erlotinib in humans. Pharmacokinetic parameter

Baseline

Rifampicin

Percentage change

AUCblood (SUV x min)

77.2 ± 19.8

86.4 ± 27.1

+11 ± 15%

AUCliver (SUV x min)

1,101 ± 310

924 ± 209 *

-14 ± 14%

AUCbile duct+gall bladder (SUV x min) 6,131 ± 2,213

4,091 ± 2,017

-15 ± 80%

AUCliver/AUCblood

15.2 ± 5.7

11.9 ± 5.7 *

-22 ± 15%

kuptake,liver (mL/min/g liver)

0.43 ± 0.12

0.35 ± 0.09 *

-18 ± 10%

K1 (mL/min/g liver)

0.94 ± 0.23 (10) 0.77 ± 0.22 (7) ** -19 ± 9%

k2 (1/min)

0.21 ± 0.11 (23) 0.16 ± 0.11 (14)

-21 ± 32%

k3 (1/min)

0.12 ± 0.12 (24) 0.06 ± 0.04 (23)

-34 ± 43%

kuptake,kidney (mL/min/g kidney)

0.51 ± 0.09

0.49 ± 0.12

-3 ± 20%

CLbile,blood (mL/min/kg)

2.1 ± 1.2

1.6 ± 1.2

-9 ± 58%

CLbile,liver (mL/min/kg)

0.11 ± 0.04

0.14 ± 0.06

+28 ± 62%

CLrenal,blood (mL/min/kg)

0.08 ± 0.03

0.21 ± 0.05 **

+219 ± 191%

CLrenal,kidney (mL/min/kg)

0.04 ± 0.02

0.10 ± 0.03 *

+224 ± 200%

Each value represents mean ± SD (n = 6, except for CLrenal,blood where n = 5). Value in parentheses represents the precision of parameter estimates expressed as mean coefficient of variation (%). * P < 0.05, ** P < 0.01, 2-sided paired t-test for comparison with baseline scan. AUC, area under the time-activity curve; kuptake,liver and K1, rate constants for transfer of radioactivity from blood into the liver estimated with integration plot analysis and

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kinetic modeling, respectively; k2, rate constant for backflux of radioactivity from liver into blood; k3, rate constant for biliary secretion of radioactivity; kuptake,kidney, rate constant for transfer of radioactivity from blood into the kidney estimated with integration plot analysis; CLbile,blood and CLbile,liver, biliary secretion clearance of radioactivity with respect to the blood or liver concentration; CLrenal,blood and CLrenal,kidney, renal clearance of radioactivity with respect to the blood or kidney concentration.

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Table 2. Pharmacokinetic parameters of [11C]erlotinib in FVB mice. Pharmacokinetic parameter

Vehicle

Rifampicin

AUCblood (SUV x min) a

46.9 ± 5.0

158.2 ± 29.8 *** +237%

AUCliver (SUV x min)

163 ± 15

316 ± 27 ***

+94%

AUCbile duct+gall bladder (SUV x min) 160 ± 20

250 ± 44 **

+56%

AUCliver/AUCblood

3.5 ± 0.3

2.0 ± 0.3 ***

-41%

kuptake,liver (mL/min/g liver)

0.68 ± 0.08

0.26 ± 0.05 ***

-62%

kbile (1/min)

0.019 ± 0.004 0.009 ± 0.004 ** -52%

kuptake,kidney (mL/min/g kidney)

0.40 ± 0.04

0.23 ± 0.10 **

-43%

CLbile,blood (mL/min/kg)

9.0 ± 1.4

4.0 ± 1.4 ***

-56%

CLrenal,blood (mL/min/kg)

0.38 ± 0.20

0.02 ± 0.01 **

-95%

Percentage change

Each value represents mean ± SD (n = 5). ** P < 0.01, *** P < 0.001, 2-sided t-test for comparison with vehicle group. a

image-derived (from the left ventricle of the heart)

AUC, area under the time-activity curve; kuptake,liver and kuptake,kidney, rate constants for transfer of radioactivity from blood into the liver and kidney, respectively, estimated with integration plot analysis; kbile, rate constant for biliary secretion of radioactivity estimated with integration plot analysis; CLbile,blood, biliary secretion clearance of radioactivity with respect to the blood concentration; CLrenal,blood, renal clearance of radioactivity with respect to the blood concentration.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 1 78x55mm (300 x 300 DPI)


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Figure 3 103x76mm (300 x 300 DPI)


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Figure 4 160x295mm (300 x 300 DPI)





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Table of contents graphic 35x16mm (300 x 300 DPI)


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Effect of Rifampicin on the Distribution of [11C ... - ACS Publications

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