Nicolas Tournier

Bruno Stieger

Dr Oliver Langer
email: oliver.langer@meduniwien.ac.at

Imaging techniques to study drug transporter function in vivo

Keywords: Imaging drug transporter function ABC, ATP-binding cassette, AD, Alzheimer's disease, ADME, Absorption, Distribution, Metabolism and Elimination, ATP, adenosine triphosphate, AUC, area under the curve, BBB, blood-brain barrier, BCRP, breast cancer resistance protein, BPND, non-displaceable binding potential, CL, total clearance, CT, computed tomography, CYP, cytochrome P450, DDI, drugdrug interaction, K1, influx rate constant, k2, efflux rate constant, MAG3, mercaptoacetyltriglycine, MATE, multidrug and toxin extrusion protein, MDR, multidrug resistance, MRI, magnetic resonance imaging, MRP, multidrug resistance-associated protein, OAT, organic anion transporter, OATP, organic anion-transporting polypeptide, OCT, organic cation transporter, OCTN, organic cation / carnitine transporter, P-gp, P-glycoprotein, PBPK, physiologically based pharmacokinetic, PET, positron emission tomography, PK, pharmacokinetics, SLC, solute carrier, SNP, single nucleotide polymorphism, SPECT, single photon emission computed tomography, TKI, tyrosine kinase inhibitor, VT, total volume of distribution Pharmacokinetics, molecular imaging, membrane transporters, positron emission tomography, magnetic resonance imaging, single photon emission computed tomography

Transporter systems involved in the permeation of drugs and solutes across biological membranes are recognized as key determinants of pharmacokinetics. Typically, the action of membrane transporters on drug exposure to tissues in living organisms is inferred from invasive procedures, which cannot be applied in humans. In recent years, imaging methods have greatly progressed in terms of instruments, synthesis of novel imaging probes as well as tools for data analysis. Imaging allows pharmacokinetic parameters in different tissues and organs to be obtained in a noninvasive or minimally invasive way. The aim of this overview is to summarize the current status in the field of molecular imaging of drug transporters. The overview is focused on human studies, both for the characterization of transport systems for imaging agents as well as for the determination of drug pharmacokinetics, and makes reference to animal studies where necessary.

We conclude that despite certain methodological limitations, imaging has a great potential to study transporters at work in humans and that imaging will become an important tool, not only in drug development but also in medicine. Imaging allows the mechanistic aspects of transport proteins to be studied, as well as elucidating the influence of genetic background, pathophysiological states and drug-drug interactions on the function of transporters involved in the disposition of drugs.

Introduction

The pharmacologic effect of drugs is directly dependent on the local concentration of the drug at its respective target and hence critically depends on the pharmacokinetics (PK) of the drug [START_REF] Fung | Perspectives on the history and scientific contributions of Gerhard Levy[END_REF][START_REF] Levy | Kinetics of pharmacologic effects[END_REF]. PK includes determining the absorption, distribution, metabolism and excretion (ADME) of a drug or xenobiotic, and describes the concentration-time profile, typically determined in the circulation [START_REF] Fan | Pharmacokinetics[END_REF]. It is evident that sampling drugs in blood plasma does not give direct and accurate information on the concentration of a drug at its target, which very often is located in tissues, outside of the circulation. Current clinical pharmacokinetic studies do not provide information on the penetration of drugs into tissues or across barriers, e.g. the blood-brain barrier (BBB), placenta or tumor barriers, to name a few. ADME of drugs and xenobiotics is governed by many factors. Crossing of plasma membranes or other biological membranes by drugs and other solutes requires in general proteins, i.e. transport systems [START_REF] Kell | What would be the observable consequences if phospholipid bilayer diffusion of drugs into cells is negligible[END_REF]. (Drug) transporters can be divided very generally into systems mediating the uptake of substances into cells and systems mediating the efflux of substances from cells [START_REF] Hediger | The ABCs of membrane transporters in health and disease (SLC series): introduction[END_REF]. Uptake of solutes into cells is largely mediated by members of the solute carrier (SLC) gene series [START_REF] Hediger | The ABCs of membrane transporters in health and disease (SLC series): introduction[END_REF]. Export may be mediated by SLC transporters or, if against steep concentration gradients, by members of the group of adenosine triphosphate (ATP)-binding cassette (ABC) transporters. Once in a cell, many drugs and solutes are metabolized. Metabolism will affect both the total and intracellular concentration of transporter substrates. The interplay of transport into cells and intracellular metabolism generates a dilemma in the elucidation of molecular mechanisms involved in drug metabolism and elimination [START_REF] Benet | The drug transporter-metabolism alliance: uncovering and defining the interplay[END_REF].

Current methodology for measuring drug concentrations in tissues requires invasive procedures [START_REF] Chu | Intracellular Drug Concentrations and Transporters: Measurement, Modeling, and Implications for the Liver[END_REF]. In such situations, imaging methods come into play, as they allow molecules in the body to be visualized in a non-invasive manner and even more so as they allow detailed information to be obtained on time-dependent changes in drug concentrations in organs or tissues.

In parallel to the acceptance of transporters as the key protein systems allowing the crossing of solutes over biological membranes [START_REF] Giacomini | Membrane transporters in drug development[END_REF][START_REF] Hillgren | Emerging transporters of clinical importance: an update from the International Transporter Consortium[END_REF][START_REF] Suzuki | Transport of drugs across the hepatic sinusoidal membrane: sinusoidal drug influx and efflux in the liver[END_REF][START_REF] Van Montfoort | Drug uptake systems in liver and kidney[END_REF], the role of drug transporters in imaging is increasingly being appreciated [START_REF] Kannan | Imaging the function of P-glycoprotein with radiotracers: pharmacokinetics and in vivo applications[END_REF][START_REF] Kilbourn | Small Molecule PET Tracers for Transporter Imaging[END_REF][START_REF] Kusuhara | Imaging in the study of membrane transporters[END_REF][START_REF] Langer | Use of PET Imaging to Evaluate Transporter-Mediated Drug-Drug Interactions[END_REF][START_REF] Mann | Imaging transporters: Transforming diagnostic and therapeutic development[END_REF][START_REF] Marie | Imaging Probes and Modalities for the Study of Solute Carrier O (SLCO)-Transport Function In Vivo[END_REF][START_REF] Stieger | The emerging role of transport systems in liver function tests[END_REF][START_REF] Testa | PET Tracers To Study Clinically Relevant Hepatic Transporters[END_REF][START_REF] Van Beers | Primovist, Eovist: what to expect[END_REF]. Noninvasive imaging methods have therefore been developed to reveal and quantify drug transporter function in clearance and non-clearance organs, as a prerequisite to the local pharmacologic/toxicological effect, metabolism and elimination. The aim of this review is to provide an overview of the application of imaging methods dedicated to the study of drug transporter function in vivo.

Imaging methods for in vivo pharmacokinetics

In vivo imaging modalities

Current research in the field of translational imaging of drug transporters is predominantly based on clinically feasible imaging approaches. This includes nuclear imaging such as single photon emission computed tomography (SPECT) or positron emission tomography (PET) as well as magnetic resonance imaging (MRI). All of these approaches rely on the use of drug transporter substrates, which are detected using appropriate imaging modalities. The main advantage of these imaging techniques is their non-invasive or minimally-invasive nature. This allows a repetitive study of the same living subject, including humans, in identical or comparative conditions at different time points, thus harnessing the statistical power of longitudinal studies and reducing the number of individuals required [START_REF] Willmann | Molecular imaging in drug development[END_REF]. This contrasts with the more conventional destructive biodistribution approaches, in which animals are sacrificed at predefined time points. In this framework, molecular imaging benefits from suitable temporal resolution to address the impact of carrier-mediated transport systems on the kinetics of drug disposition in vivo [START_REF] Cunha | The role of molecular imaging in modern drug development[END_REF].

Scintigraphy captures emitted radiation from administered gamma-emitting radioisotopes to create two-dimensional and poorly quantitative "planar" acquisitions. Tomographic acquisitions and computed tomography reconstruction (i.e. SPECT) aim at overcoming this limitation by providing 3D images of the distribution of the signal. Usually one, two or three detectors are slowly rotated around the body, leading to a considerable loss in time-resolution [START_REF] Frey | Accuracy and precision of radioactivity quantification in nuclear medicine images[END_REF].

SPECT has traditionally been considered as a non-quantitative imaging method, but recent advances in medical physics have now made SPECT a quantitative method due to coupling with a computed tomography (CT) scan in hybrid SPECT/CT scanners. Quantification with clinical SPECT/CT scanners, however, remains of slightly lower performance than with PET, especially in small volume regions, due to marginally poorer image spatial resolution (typically ~7-10 mm for SPECT versus ~5-8 mm for PET) [START_REF] Bailey | An Evidence-Based Review of Quantitative SPECT Imaging and Potential Clinical Applications[END_REF]. SPECT benefits from good sensitivity for the detection of radioactivity, allowing for the use of imaging agents at tracer doses with a limited risk of toxicity. Despite its technical limitations, SPECT represents a relevant and cost-effective tool for the study of transporter function in a conventional hospital environment (Fig. 1) [START_REF] Willmann | Molecular imaging in drug development[END_REF].

PET uses radioactive isotopes emitting positrons (ß + decay), which collide with electrons in the tissue. Subsequent annihilation of these particles releases two photons, which can be detected and allow calculation of the point where the positron and the electron collided. Today, numerous PET tracers are used in medical practice [START_REF] Vallabhajosula | A broad overview of positron emission tomography radiopharmaceuticals and clinical applications: what is new?[END_REF]. PET imaging has also been used for decades in drug development [START_REF] Willmann | Molecular imaging in drug development[END_REF]. The advantages of PET over SPECT include higher sensitivity (detection efficiency) and better temporal and spatial resolution.

PET imaging benefits from straightforward 3D quantifiable recordings. Owing to the high sensitivity of PET scanners, radiopharmaceutical agents used for PET imaging can be administered at tracer doses, usually in the range of a few micrograms (Fig. 1). PET is a functional imaging technique which only gives limited anatomical information. For the delineation of organ substructures on PET images, anatomical information is often required. This can be obtained either by performing additional scans on stand-alone MRI or CT scanners and co-registration of the data from the two imaging modalities, or by using integrated imaging systems (PET/CT, PET/MRI), which provide both functional and anatomical information in a single scanning session [START_REF] Beyer | A combined PET/CT scanner for clinical oncology[END_REF][START_REF] Beyer | PET/MRI-knocking on the doors of the rich and famous[END_REF].

Compared with nuclear imaging techniques, MRI is a non-irradiant imaging modality. MRI measures the energy released in the form of a radiofrequency of atoms, which realign to a strong magnetic field after being forced out of this alignment, and requires atomic nuclei with an odd number of protons and/or neutrons. MRI produces pictures of anatomy and of physiological processes with high spatial resolution and excellent soft tissue contrast. Several MRI contrast agents are now available, including paramagnetic gadolinium compounds [START_REF] Xiao | MRI contrast agents: Classification and application (Review)[END_REF].

Compared with nuclear imaging, which can be performed using tracer doses of radiopharmaceutical agents, the contrast agents used for MRI must be administered at pharmacologic doses in order to be detected. The sensitivity is thus limited (micro-to millimolar) and quantification of the absolute concentration of the contrast agent is challenging [START_REF] Sinharay | Advances in Magnetic Resonance Imaging Contrast Agents for Biomarker Detection[END_REF]. MRI is nonetheless able to capture the rapid temporal signal changes (contrast enhancement) associated with the presence of a contrast agent in tissues. MRI thus offers semiquantitative data derived from high temporal and spatial resolution images that are of interest for studying transporter function in vivo [START_REF] Georgiou | Quantitative Assessment of Liver Function Using Gadoxetate-Enhanced Magnetic Resonance Imaging[END_REF] (Fig. 1).

Chemistry of imaging probes

To date, transporter imaging probes are usually not molecules designed de novo, but are mostly derived from known drug molecules, drug metabolites or from endogenous compounds (e.g. bile acids) [START_REF] Kusuhara | Imaging in the study of membrane transporters[END_REF][START_REF] Mairinger | PET and SPECT radiotracers to assess function and expression of ABC transporters in vivo[END_REF][START_REF] Testa | PET Tracers To Study Clinically Relevant Hepatic Transporters[END_REF]. The classical positron-emitting radionuclides for PET imaging are carbon-11 ( 11 C, half-life: 20.4 min) and fluorine-18 ( 18 F, halflife: 109.7 min). These radionuclides are produced in a cyclotron, and the synthesis of PET radiotracers is typically performed immediately before administration in dedicated lead-shielded automated synthesis modules. Today, the number of specialized PET centers is limited, which limits the applicability of this technique [START_REF] Willmann | Molecular imaging in drug development[END_REF] (Fig. 1).

11 C is the preferred PET radionuclide as it allows radiolabeling of drugs without structural modifications. Due to the versatility of PET radiochemistry, PET imaging benefits from a large portfolio of available radiotracers when compared with other clinical imaging modalities (Fig. 1).

11 C-labeling is most commonly performed by 11 C-methylation of phenolic hydroxyl, carboxylic acid or amino groups (Fig. 2). Recent alternative approaches to 11 C-labeling have been developed, thus giving the possibility of labeling a wider range of chemical structures with 11 C [START_REF] Li | Radiopharmaceutical chemistry for positron emission tomography[END_REF]. For drugs which contain fluorine, 18 F may be employed for radiolabeling [START_REF] Wuest | Fluorine-18 labeling of small molecules: the use of 18F-labeled aryl fluorides derived from no-carrier-added [18F]fluoride as labeling precursors[END_REF].

The latter offers the advantage of a longer radioactive half-life than that of 11 C, thereby enabling the measurement of drug tissue PK over several hours [START_REF] Brunner | Ciprofloxacin, a new positron emission tomography tracer for noninvasive assessment of the tissue distribution and pharmacokinetics of ciprofloxacin in humans[END_REF]. A typical 18 F-labeling reaction is the nucleophilic substitution reaction with [ 18 F]fluoride of suitable leaving groups (Fig. 2). PET radiochemistry is usually performed at high molar activity (molar activity = ratio of radioactivity to mass, given for instance as GBq/µmol). Therefore, the chemical mass of unlabeled drug contained in a typically injected amount of a PET tracer (approximately 400 MBq) is very low (< 100 µg). This satisfies the definition of a microdose set forth by regulatory authorities, which requires less extensive preclinical toxicology testing for applications in humans than is required for standard phase one clinical trials, thereby facilitating first-in-human studies (ICH Expert Working Group, 2009).

For SPECT radiochemistry, typical radionuclides for the radiolabeling of small molecules are technetium-99m ( 99m Tc, half-life, 6.1 h) and iodine-123 ( 123 I, half-life: 13.3 h). 99m Tc allows for clinical grade and kit-based preparation of radiopharmaceutical agents for human use and often provides labels with suitable metabolic stability in vivo. Several 99m Tc-labeled imaging agents used in the clinics have been re-purposed as probes for molecular imaging of drug transporters [START_REF] Kusuhara | Imaging in the study of membrane transporters[END_REF].

The variety of available contrast agents for MRI is very limited. MRI contrast agents predominantly contain metals (mostly the transition metal gadolinium) associated by complex chemistry. MRI is increasingly considered as a molecular imaging method owing to the availability of contrast agents to detect molecular processes at the tissue level [START_REF] Sinharay | Advances in Magnetic Resonance Imaging Contrast Agents for Biomarker Detection[END_REF]. Gadoxetate (gadolinium-ethoxybenzyl-diethylenetriamine-pentaacetic acid, Gd-EOB-DTPA, Fig. 2) is a MRI contrast agent which induces liver-specific contrast enhancement due to transporter-mediated uptake into hepatocytes and biliary secretion of gadoxetate [START_REF] Van Montfoort | Hepatic uptake of the magnetic resonance imaging contrast agent gadoxetate by the organic anion transporting polypeptide Oatp1[END_REF]. This contrast agent benefits from a suitable complex stability in vivo without any apparent biotransformation, making it suitable to study liver transporter function in vivo.

Modeling transporter function in vivo

Of the available imaging techniques, PET is the preferred modality for kinetic modeling purposes as it provides fully quantitative tissue concentrations of radioactivity (Fig. 1). For dynamic contrastenhanced MRI, conversion of contrast enhancement in tissue into absolute concentration levels of contrast agent is feasible but challenging [START_REF] Ingrisch | Tracer-kinetic modeling of dynamic contrast-enhanced MRI and CT: a primer[END_REF][START_REF] Sourbron | Classic models for dynamic contrast-enhanced MRI[END_REF]. When the time course of radiotracer in arterial blood (the so-called arterial input function) is also known, compartmental modeling approaches can be employed to derive pharmacokinetic parameters describing the tissue distribution of radiotracer. The modeling approaches used for analysis of imaging data are conceptually similar to modeling approaches used in the pharmaceutical sciences, but they employ different terminology [START_REF] Innis | Consensus nomenclature for in vivo imaging of reversibly binding radioligands[END_REF]. The gold standard to measure the arterial input function is arterial blood sampling. Pharmacokinetic modeling of imaging data can, for instance, provide the exchange rate constants of radiotracer between plasma and tissue compartments as outcome parameters [START_REF] Innis | Consensus nomenclature for in vivo imaging of reversibly binding radioligands[END_REF]. As ABC and SLC transporters are localized at blood-tissue interfaces, these exchange rate constants have been identified as the key parameters reflecting transporter activities. For example, the activity of efflux transporters at the BBB, such as P-glycoprotein (P-gp, ABCB1) or breast cancer resistance protein (BCRP, ABCG2), was shown to mainly affect the transfer rate constant of radiotracer from plasma into brain (K1). A reduction in efflux transporter activity at the BBB (e.g. by administration of a transporter inhibitor) was shown to increase the K1 value [START_REF] Muzi | Imaging of cyclosporine inhibition of P-glycoprotein activity using 11C-verapamil in the brain: studies of healthy humans[END_REF][START_REF] Wagner | A pilot study to assess the efficacy of tariquidar to inhibit P-glycoprotein at the human blood-brain barrier with (R)-11C-verapamil and PET[END_REF]. Conceptually, efflux transporters at the BBB should also affect the efflux rate constant from brain to plasma (k2) [START_REF] Syvänen | Pharmacokinetic consequences of active drug efflux at the blood-brain barrier[END_REF], which was indeed observed for certain compounds [START_REF] Pottier | Imaging the Impact of the P-Glycoprotein (ABCB1) Function on the Brain Kinetics of Metoclopramide[END_REF]. Inhibition of an uptake transporter in the sinusoidal membrane of hepatocytes will lead to a reduction in the transfer rate constant or in the uptake clearance from blood into liver (CLuptake,liver), which may be determined with graphical analysis approaches (integration plot analysis) [START_REF] Takashima | PET imaging-based evaluation of hepatobiliary transport in humans with (15R)-11C-TIC-Me[END_REF]. Integration plot analysis has also been used to obtain intrinsic efflux clearances of radiotracers from liver into bile (CLint,bile) or from kidney into urine (CLint,urine) to measure the activities of efflux transporters located at the canalicular membrane of hepatocytes or at the brush border membrane of kidney proximal tubule cells, respectively [START_REF] Shingaki | Quantitative Evaluation of mMate1 Function Based on Minimally Invasive Measurement of Tissue Concentration Using PET with [(11)C]Metformin in Mouse[END_REF][START_REF] Takashima | Evaluation of breast cancer resistance protein function in hepatobiliary and renal excretion using PET with 11C-SC-62807[END_REF][START_REF] Traxl | Hepatocyte-Specific Deletion of EGFR in Mice Reduces Hepatic Abcg2 Transport Activity Measured by [(11)C]erlotinib and Positron Emission Tomography[END_REF].

Depending on the organ investigated, modeling approaches can become very complex. For instance, the modeling of liver data is complicated by the fact that the liver receives dual blood supply, via the hepatic artery (~ 25%) and via the portal vein (~ 75%). Portal blood cannot be directly sampled in humans. Therefore, modeling approaches either have to rely on image-derived portal blood curves, which may be prone to imaging artefacts due to the small size of the investigated structures, or on data acquired in preclinical species (e.g. pigs) in which portal blood can be sampled [START_REF] Ørntoft | Hepatobiliary transport kinetics of the conjugated bile acid tracer 11C-CSar quantified in healthy humans and patients by positron emission tomography[END_REF][START_REF] Sørensen | Hepatobiliary Secretion Kinetics of Conjugated Bile Acids Measured in Pigs by 11C-Cholylsarcosine PET[END_REF]. Figure 3 shows a kinetic model used for quantification of hepatic disposition of the radiolabeled bile acid [ 11 C]cholylsarcosine in humans (Ørntoft et al., 2017). Similar kinetic modeling approaches to those used for the analysis of PET data have also been applied to SPECT data [START_REF] Pfeifer | Effect of Ritonavir on (99m)Technetium-Mebrofenin Disposition in Humans: A Semi-PBPK Modeling and In Vitro Approach to Predict Transporter-Mediated DDIs[END_REF][START_REF] Neyt | In vivo visualization and quantification of (Disturbed) Oatp-mediated hepatic uptake and Mrp2mediated biliary excretion of 99mTc-mebrofenin in mice[END_REF] and to MRI data [START_REF] Sourbron | Combined quantification of liver perfusion and function with dynamic gadoxetic acid-enhanced MR imaging[END_REF][START_REF] Saito | Assessing liver function using dynamic Gd-EOB-DTPA-enhanced MRI with a standard 5-phase imaging protocol[END_REF].

Metabolism of imaging probes

Like most xenobiotics, imaging probes may often undergo significant in vivo biotransformation.

Paradoxically, "molecular imaging" techniques are devoid of "molecular resolution". Nuclear imaging techniques (PET, SPECT) can only measure total radioactivity in tissues. Radioactivity originating from radiolabeled parent drug cannot be distinguished from radioactivity originating from radiolabeled metabolites [START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF]. While metal chelates used as probes to study drug transporters with SPECT and MRI often benefit from good in vivo stability, 11 C-and 18 F-labeled small, drug-like molecules used in PET studies often undergo extensive metabolism [START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF].

The study of drug transporters with radiotracers which are extensively metabolized can provide misleading results, in that changes in radiotracer metabolism can be erroneously interpreted as changes in transporter activity. If circulating radiolabeled metabolites are present in blood, total radioactivity counts in blood samples can be corrected for these radiolabeled metabolites using ex vivo chromatographic methods. On the other hand, radiolabeled metabolites in tissues cannot be corrected for and will inevitably contribute to the imaging signal. During radiotracer development, tissue-penetrant radiometabolites can be detected and identified in tissue lysates obtained from terminal experiments in rodents [START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF]. However, species differences in radiotracer metabolism may lead to the presence of tissue-penetrant radiolabeled metabolites in humans where not observed in rodents, or vice versa. The contribution of radiolabeled metabolites to the tissue PET signal in humans can only be indirectly inferred, for instance by the observation that pharmacokinetic modeling yields results which are inconsistent with the presence of a single radioactive species. The best approach to assess the contribution of radiolabeled metabolites to the PET signal in humans would be to directly radiolabel the metabolite of interest for PET imaging experiments, which is, however, very labor intensive and therefore rarely done. However, according to the authors' own experience, PET tracers based on known drug molecules which are mainly excreted in the form of metabolites may undergo a relatively low degree of metabolism over the short duration of an imaging experiment (e.g. 60-90 min for [ 11 C]radiotracers), so that they may still be useful to study transporter function. One such example is erlotinib, for which mass balance studies in humans indicated that only < 2% of total radioactivity excreted into feces was composed of unmetabolized parent compound [START_REF] Ling | Metabolism and excretion of erlotinib, a small molecule inhibitor of epidermal growth factor receptor tyrosine kinase, in healthy male volunteers[END_REF]. PET experiments with [ 11 C]erlotinib in mice, on the other hand, indicated that at 25 min after radiotracer injection the majority of radioactivity in plasma and different tissues and fluids was in the form of unmetabolized [ 11 C]erlotinib [START_REF] Traxl | Breast Cancer Resistance Protein and P-Glycoprotein Influence In Vivo Disposition of 11C-Erlotinib[END_REF], suggesting that this radiotracer can be used to study transporter function. One approach to overcome the limitation of radiotracer metabolism may be the radiolabeling of drug metabolites and their use as transporter imaging probes with better metabolic stability, as exemplified by the P-gp imaging probe [ 11 C]N-desmethyl-loperamide [START_REF] Lazarova | Synthesis and evaluation of [N-methyl-11C]N-desmethyl-loperamide as a new and improved PET radiotracer for imaging P-gp function[END_REF] or by the BCRP imaging probe [ 11 C]SC-62807 [START_REF] Takashima | Evaluation of breast cancer resistance protein function in hepatobiliary and renal excretion using PET with 11C-SC-62807[END_REF]. Moreover, the problem of radiolabeled metabolites can be overcome by performing data analysis over a short and early time window after radiotracer injection in which radiotracer metabolism is negligible. For instance, the P-gp probe substrate [ 11 C]verapamil is extensively metabolized in vivo, generating radiolabeled metabolites which are also P-gp substrates and which are taken up from periphery into the brain [START_REF] Luurtsema | Evaluation of (R)-[11C]verapamil as PET tracer of P-glycoprotein function in the blood-brain barrier: kinetics and metabolism in the rat[END_REF]. Some investigators have therefore used data from the first few minutes after radiotracer injection only, when [ 11 C]verapamil accounts for more than 80% of the plasma radioactivity, to determine K1 as a parameter of P-gp function at the BBB [START_REF] Ikoma | Quantitative analysis of 11C-verapamil transfer at the human blood-brain barrier for evaluation of P-glycoprotein function[END_REF] [START_REF] Muzi | Imaging of cyclosporine inhibition of P-glycoprotein activity using 11C-verapamil in the brain: studies of healthy humans[END_REF]. Similarly, integration plot analysis to determine the uptake clearance of radiotracers from blood into the liver usually relies exclusively on data from the first few minutes after radiotracer injection, where the contribution of radiolabeled metabolites can often be neglected [START_REF] Kaneko | A clinical quantitative evaluation of hepatobiliary transport of [11C]Dehydropravastatin in humans using positron emission tomography[END_REF]. For the analysis of apical ABC efflux transporters in the liver or kidney, on the other hand, good metabolic stability of the PET tracer is mandatory as metabolism may otherwise confound the determination of CLint,bile or CLint,urine values.

Imaging probes to study transporter function

Liver transporters

The liver is a key organ involved in the metabolism and excretion of xenobiotics. Consequently, the expression of a large variety of transporters has been observed in this organ and the role of some of these transporters is well established in pharmacokinetic drug-drug interactions (DDIs) and in adverse drug actions in the liver. Organic anion-transporting polypeptides (OATPs) have a very broad substrate specificity and are key transporters for drugs entering hepatocytes [START_REF] Hagenbuch | The SLCO (former SLC21) superfamily of transporters[END_REF]. In addition, OATPs are known to mediate the transmembrane transport of a considerable number of imaging agents [START_REF] Marie | Imaging Probes and Modalities for the Study of Solute Carrier O (SLCO)-Transport Function In Vivo[END_REF]. The founding members of the SLCO gene superfamily encoding OATPs were shown to transport the MRI contrast agent gadoxetate [START_REF] Van Montfoort | Hepatic uptake of the magnetic resonance imaging contrast agent gadoxetate by the organic anion transporting polypeptide Oatp1[END_REF] (Fig. 2). The SPECT tracer [ 99m Tc]mebrofenin (Fig. 2) is likewise taken up into the liver via OATPs and can be used for hepatobiliary imaging [START_REF] De Graaf | Transporters involved in the hepatic uptake of (99m)Tc-mebrofenin and indocyanine green[END_REF][START_REF] Ghibellini | Use of tc-99m mebrofenin as a clinical probe to assess altered hepatobiliary transport: integration of in vitro, pharmacokinetic modeling, and simulation studies[END_REF]. However, transport of most hepatic imaging agents is not restricted to OATPs but may also include organic anion transporters (OATs), organic cation transporters (OCTs) and ABC transporters [START_REF] Kilbourn | Small Molecule PET Tracers for Transporter Imaging[END_REF][START_REF] Mairinger | PET and SPECT radiotracers to assess function and expression of ABC transporters in vivo[END_REF][START_REF] Stieger | Role of (drug) transporters in imaging in health and disease[END_REF][START_REF] Testa | PET Tracers To Study Clinically Relevant Hepatic Transporters[END_REF].

The transporters for bile salts in hepatocytes have been identified and characterized at the molecular level [START_REF] Hagenbuch | The SLCO (former SLC21) superfamily of transporters[END_REF][START_REF] Stieger | The role of the sodium-taurocholate cotransporting polypeptide (NTCP) and of the bile salt export pump (BSEP) in physiology and pathophysiology of bile formation[END_REF]. Bile salt derivatives are excellent target molecules for developing imaging agents to study liver transporters of importance in PK.

For example, cholylsarcosine is a synthetic bile acid which is resistant to deconjugation and dehydroxylation [START_REF] Schmassmann | Transport, metabolism, and effect of chronic feeding of cholylsarcosine, a conjugated bile acid resistant to deconjugation and dehydroxylation[END_REF]. A 11 C-labeled version of this bile acid was successfully used to visualize in vivo the kinetics of bile acid secretion in pigs (Frisch et al., 2012;[START_REF] Sørensen | Hepatobiliary Secretion Kinetics of Conjugated Bile Acids Measured in Pigs by 11C-Cholylsarcosine PET[END_REF]. In a recent proof-of-concept study, this bile acid was found to be a suitable probe to non-invasively assess bile flow in humans with liver disease (Ørntoft et al., 2017). In addition, the successful synthesis of 11 C-labeled derivatives of taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, tauroursodeoxycholic acid and taurolithocholic acid was reported [START_REF] Schacht | Radiosynthesis of N-11 C-Methyl-Taurine-Conjugated Bile Acids and Biodistribution Studies in Pigs by PET/CT[END_REF]. All derivatives display comparable biodistribution in pigs, and enterohepatic circulation of the taurocholate derivative could be observed. Using a different synthetic strategy, an 18 F-labeled chenodeoxycholic derivative was developed and demonstrated by PET imaging to be concentrated in the liver of mice in a timedependent manner [START_REF] Jia | Synthesis and evaluation of (18)F-labeled bile acid compound: a potential PET imaging agent for FXR-related diseases[END_REF]. However, as the click chemistry-based 18 F-labeling reaction led to modification of the carboxyl group, some concerns were raised as to whether this tracer was handled by the same hepatic transporters as endogenous bile acids [START_REF] Frisch | On fluoro-18 labeling of bile acids[END_REF].

Recently, an 18 F-labeled derivative of unconjugated cholic acid (3 -[ 18 F]fluorocholic acid, Fig. 2) was shown to be a substrate of Na + -taurocholate co-transporting polypeptide (NTCP, SLC10A1), OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) [START_REF] De Lombaerde | Synthesis, in vitro and in vivo evaluation of 3β-[18F]fluorocholic acid for the detection of drug-induced cholestasis in mice[END_REF], all of which are known to be involved in hepatocellular bile acid and bile salt uptake [START_REF] Stieger | The role of the sodium-taurocholate cotransporting polypeptide (NTCP) and of the bile salt export pump (BSEP) in physiology and pathophysiology of bile formation[END_REF]. Furthermore, this bile acid derivative inhibited bile salt export pump (BSEP, ABCB11)-mediated taurocholate and MRP2-mediated estradiol-17ß-glucuronide transport. In mice, pretreatment with rifampicin or bosentan [START_REF] Fattinger | Rifamycin SV and rifampicin exhibit differential inhibition of the hepatic rat organic anion transporting polypeptides, Oatp1 and Oatp2[END_REF][START_REF] Fattinger | The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions[END_REF][START_REF] Stieger | Drug-and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver[END_REF] was shown to interfere with hepatocellular transport of 3 -[ 18 F]fluorocholic acid [START_REF] De Lombaerde | Synthesis, in vitro and in vivo evaluation of 3β-[18F]fluorocholic acid for the detection of drug-induced cholestasis in mice[END_REF].

In addition to studying the function of bile acid transporters, PET has been used to study ABC efflux transporter function in the canalicular membrane of hepatocytes. For instance, the radiolabeled metabolite of the COX-2 inhibitor celecoxib [ 11 C]SC-62807 was shown to undergo biliary secretion mediated by mBCRP and should thus allow measurement of BCRP function in the liver [START_REF] Takashima | Evaluation of breast cancer resistance protein function in hepatobiliary and renal excretion using PET with 11C-SC-62807[END_REF]. PET imaging with [ 11 C]dehydropravastatin allows assessment of the transport activities of OATPs and MRP2 (ABCC2) in the rat and human liver [START_REF] Kaneko | A clinical quantitative evaluation of hepatobiliary transport of [11C]Dehydropravastatin in humans using positron emission tomography[END_REF][START_REF] Shingaki | Evaluation of Oatp and Mrp2 Activities in Hepatobiliary Excretion Using Newly Developed Positron Emission Tomography Tracer [11C]Dehydropravastatin in Rats[END_REF].

MRI is one of the methods used to examine patients for presence of focal liver lesions [START_REF] Pastor | The role of organic anion transporters in diagnosing liver diseases by magnetic resonance imaging[END_REF]. MRI contrast agents are typically negatively charged [START_REF] Xiao | MRI contrast agents: Classification and application (Review)[END_REF] and are substrates of transport systems for organic anions [START_REF] Marie | Imaging Probes and Modalities for the Study of Solute Carrier O (SLCO)-Transport Function In Vivo[END_REF]. Gadoxetate was found to be transported by human OATPB1 and OATP1B3, but not by OATP2B1 (SLCO2B1) [START_REF] Leonhardt | Hepatic Uptake of the Magnetic Resonance Imaging Contrast Agent Gd-EOB-DTPA: Role of Human Organic Anion Transporters[END_REF]. In addition, gadoxetate is a weak substrate of NTCP, which may further contribute to its liver uptake [START_REF] Leonhardt | Hepatic Uptake of the Magnetic Resonance Imaging Contrast Agent Gd-EOB-DTPA: Role of Human Organic Anion Transporters[END_REF]. Gadoxetate was also shown to be transported by MRP2 (Jia et al., 2014a), which mediates its biliary secretion in rats (Jia et al., 2014a). In one study, gadoxetate was found not to be transported by OATP1A2 (SLCO1A2) [START_REF] Van Montfoort | Hepatic uptake of the magnetic resonance imaging contrast agent gadoxetate by the organic anion transporting polypeptide Oatp1[END_REF], while others identified gadoxetate as a substrate of OATP1A2 and MRP3 but not of the apical sodium-dependent bile acid transporter (ABST, SLC10A2) and OCT3 (SLC22A3) (Jia et al., 2014a). Lagadec and coworkers recently reported a correlation between the hepatic extraction fraction of gadoxetate and the expression of rOATP1A1 in a rat model of advanced liver fibrosis [START_REF] Lagadec | Advanced fibrosis: Correlation between pharmacokinetic parameters at dynamic gadoxetateenhanced MR imaging and hepatocyte organic anion transporter expression in rat liver[END_REF]. BOPTA (gadolinium benzyl-oxypropionictetraacetate) is another MRI contrast agent labeled with gadolinium. In situ perfusion of rat livers suggested that BOPTA uptake is mediated by rodent OATP transporters. In vitro experiments revealed that BOPTA was transported by rOATP1A1, rOATP1A4, rOATP1B2 and by rMRP2 [START_REF] Planchamp | Function of both sinusoidal and canalicular transporters controls the concentration of organic anions within hepatocytes[END_REF].

Kidney transporters

Renal clearance is a major pathway of drug elimination, which is the result of three concurrent processes occurring in the nephron, including glomerular filtration, tubular secretion and tubular reabsorption. Glomerular filtration is a passive process, while tubular secretion and reabsorption involve drug transporters located in the basolateral and luminal membranes of the tubular epithelium [START_REF] Yin | Renal drug transporters and their significance in drug-drug interactions[END_REF]. In humans, the major transporters involved in tubular secretion of drugs include OCT2 (SLC22A2), OAT1 (SLC22A6), OAT3 (SLC22A8) and OATP4C1 (SLCO4C1) in the basolateral membrane and the multidrug and toxin extrusion (MATE) proteins MATE1 (SLC47A1) and MATE2-K (SLC47A2), P-gp, MRP2 and MRP4 (ABCC4) in the apical membrane [START_REF] Giacomini | Membrane transporters in drug development[END_REF]. Several other related transporters are present in the proximal tubules and may also play a role in the renal elimination of drugs and drug metabolites [START_REF] Yin | Renal drug transporters and their significance in drug-drug interactions[END_REF].

Radionuclide imaging of the kidneys aims at detecting the pathophysiology of the diseased kidneys and at achieving an early and sensitive diagnosis [START_REF] Szabo | Molecular imaging of the kidneys[END_REF]. Hippurate is a toxic urate uremic toxin that accumulates during chronic renal failure. In the past, the renal secretion of the exogenous aryl amine derivatives para-aminohippurate and the radiolabeled O-[ 131 I]iodohippurate were used as markers of tubular function in patients [START_REF] Lowenstein | The rebirth of interest in renal tubular function[END_REF]. OAT1 was shown to be the main transporter for para-aminohippurate at the proximal tubule [START_REF] Vallon | Overlapping in vitro and in vivo specificities of the organic anion transporters OAT1 and OAT3 for loop and thiazide diuretics[END_REF]. Radiolabeled derivatives of hippurate such as p-[ 18 F]fluorohippurate have been proposed as PET radioligands with improved imaging performance [START_REF] Awasthi | Synthesis and in vivo evaluation of p-18F-Fluorohippurate as a new radiopharmaceutical for assessment of renal function by PET[END_REF].

Today, [ 99m Tc]MAG3 (mercaptoacetyltriglycine) clearance from kidneys is used in clinics to investigate tubular function in patients [START_REF] Taylor | Radionuclides in Nephrourology, Part 1: Radiopharmaceuticals, Quality Control, and Quantitative Indices[END_REF]. [ 99m Tc]MAG3 is highly protein-bound and was shown to be cleared from plasma primarily by OAT1 in the kidneys [START_REF] Shikano | Transport of 99mTc-MAG3 via rat renal organic anion transporter 1[END_REF].

Furosemide, probenecid and ibuprofen were shown to be competitive inhibitors of renal OAT1 which impact [ 99m Tc]MAG3 renography [START_REF] Szabo | Molecular imaging of the kidneys[END_REF]. Renal secretion of the P-gp substrate [ 99m Tc]sestamibi was shown to be inhibited by PSC833, a cyclosporine A derivative developed as a P-gp inhibitor [START_REF] Luker | Modulation of the multidrug resistance P-glycoprotein: detection with technetium-99m-sestamibi in vivo[END_REF].

Several PET radiotracers have been developed to investigate other specific transport systems in the kidneys. In the kidney, metformin is transported into the proximal tubule via OCT2 and excreted into the urine via MATE1 and MATE2-K [START_REF] Hume | The synthesis and biodistribution of [11C]metformin as a PET probe to study hepatobiliary transport mediated by the multi-drug and toxin extrusion transporter 1 (MATE1) in vivo[END_REF]. Pedersen et al. performed [ 11 C]metformin PET imaging in a mouse model of chronic kidney disease and suggested a contribution of mOCT2 in controlling the influx rate constant of [ 11 C]metformin into the renal cortex [START_REF] Pedersen | Renal PET-imaging with (11)C-metformin in a transgenic mouse model for chronic kidney disease[END_REF]. In pigs, renal clearance of [ 11 C]metformin was approximately 3 times the glomerular filtration rate, demonstrating tubular secretion [START_REF] Jakobsen | A PET Tracer for Renal Organic Cation Transporters, 11 C-Metformin: Radiosynthesis and Preclinical Proof-of-Concept Studies[END_REF]. Takano and coworkers showed that sulpriride is a substrate of OCT1 (SLC22A1), OCT2, MATE1, and MATE2-K and proposed [ 11 C]sulpiride as a PET probe to study renal OCT function in humans [START_REF] Takano | Possible Role of Organic Cation Transporters in the Distribution of [11C]Sulpiride, a Dopamine D2Receptor Antagonist[END_REF].

Brain transporters

ABC and SLC transporters are expressed in the luminal and abluminal membranes of brain capillary endothelial cells forming the BBB, where they control access of exogenous and endogenous molecules from blood into brain parenchyma [START_REF] Abbott | Structure and function of the blood-brain barrier[END_REF][START_REF] Stieger | Drug transporters in the central nervous system[END_REF]. Among different ABC and SLC transporters expressed at the BBB, P-gp is the most widely studied transporter. Previous review articles have given detailed overviews of radiotracers for imaging of P-gp at the BBB [START_REF] Luurtsema | PET Tracers for Imaging of ABC Transporters at the Blood-Brain Barrier: Principles and Strategies[END_REF][START_REF] Mairinger | PET and SPECT radiotracers to assess function and expression of ABC transporters in vivo[END_REF][START_REF] Raaphorst | Radiopharmaceuticals for assessing ABC transporters at the blood-brain barrier[END_REF][START_REF] Wanek | Radioligands targeting P-glycoprotein and other drug efflux proteins at the blood-brain barrier[END_REF]. The applicability of the SPECT radioligand [ 99m Tc]sestamibi for imaging P-gp function at the BBB is limited due to its very low BBB permeability, even in the absence of P-gp function [START_REF] Cattelotte | Changes in dipole membrane potential at the mouse blood-brain barrier enhance the transport of 99mTechnetium Sestamibi more than inhibiting Abcb1, Abcc1, or Abcg2[END_REF]. Racemic [ 11 C]verapamil, (R)-[ 11 C]verapamil (Fig. 2) and [ 11 C]Ndesmethyl-loperamide are PET tracers which are transported by P-gp and not by BCRP [START_REF] Kannan | N-desmethyl-loperamide is selective for P-glycoprotein among three ATP-binding cassette transporters at the blood-brain barrier[END_REF][START_REF] Luurtsema | R)-and (S)-[11C]verapamil as PET-tracers for measuring P-glycoprotein function: in vitro and in vivo evaluation[END_REF][START_REF] Römermann | verapamil is selectively transported by murine and human P-glycoprotein at the blood-brain barrier, and not by MRP1 and BCRP[END_REF] and which have been used to visualize P-gp function at the rodent and human BBB in various settings. These radiotracers were very useful in measuring the effects of P-gp inhibition at the BBB with inhibitors such as cyclosporine A and tariquidar [START_REF] Bauer | Pgpmediated interaction between (R)-[11C]verapamil and tariquidar at the human blood-brain barrier: a comparison with rat data[END_REF][START_REF] Damont | Imaging the impact of cyclosporin A and dipyridamole on P-glycoprotein (ABCB1) function at the blood-brain barrier: A [(11)C]-N-desmethyl-loperamide PET study in nonhuman primates[END_REF][START_REF] Kreisl | Increased permeability-glycoprotein inhibition at the human blood-brain barrier can be safely achieved by performing PET during peak plasma concentrations of tariquidar[END_REF][START_REF] Sasongko | Imaging P-glycoprotein transport activity at the human blood-brain barrier with positron emission tomography[END_REF]. Figure 4 shows PET data on the effect of the third-generation P-gp inhibitor tariquidar on brain distribution of (R)-[ 11 C]verapamil in rats and in humans.

Beside the imaging of P-gp, efforts have also been directed towards the development of PET tracers for other ABC transporters at the BBB. Quantitative targeted absolute proteomics work indicated that BCRP is the most abundant ABC transporter at the human BBB [START_REF] Uchida | Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors[END_REF]. The development of BCRP-selective PET tracers is hindered by the overlapping substrate specificities between BCRP and P-gp. The few BCRP-selective compounds tested as PET tracers to date all possessed similar brain kinetics in wild-type and BCRP knockout mice or rats, suggesting lack of suitability to measure BCRP function at the BBB (Hosten et al., 2013;[START_REF] Mairinger | Synthesis and in vivo evaluation of the putative breast cancer resistance protein inhibitor [11C]methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-2-carbonylamino)benzoate[END_REF][START_REF] Sivapackiam | 67/68)Galmydar: A metalloprobe for monitoring breast cancer resistance protein (BCRP)mediated functional transport activity[END_REF][START_REF] Takashima | Evaluation of breast cancer resistance protein function in hepatobiliary and renal excretion using PET with 11C-SC-62807[END_REF]. Wanek and co-workers developed a PET protocol for visualization of BCRP function at the BBB based on PET scans with the dual P-gp/BCRP substrate [ 11 C]tariquidar, in which unlabeled tariquidar is co-administered to inhibit Pgp at the BBB [START_REF] Wanek | A novel PET protocol for visualization of breast cancer resistance protein function at the bloodbrain barrier[END_REF]. Okamura et al. developed 6-bromo-7-[ 11 C]methylpurine as a PET tracer to measure the function of multidrug resistance-associated protein (MRP1, ABCC1) in the brain. The PET tracer crosses the BBB, presumably by passive diffusion, and is converted inside the brain by glutathione-S-transferases into its glutathione conjugate, which leaves the brain by MRP1 efflux [START_REF] Okamura | Noninvasive and quantitative assessment of the function of multidrug resistance-associated protein 1 in the living brain[END_REF]. While this radiotracer showed great differences in brain clearance between wild-type and Abcc1 (-/-) mice, the exact site in the brain at which MRP1 contributes to radioactivity elimination (e.g. choroid plexus, glia cells or brain capillary endothelial cells) has not yet been fully established. Galante and colleagues recently synthesized a 18 Flabeled version of this MRP1 tracer [START_REF] Galante | Development of purine-derived 18F-labeled pro-drug tracers for imaging of MRP1 activity with PET[END_REF] for which brain clearance was also shown to be dependent on mMRP1 function.

In addition to efflux transporters, uptake transporters are also expressed at the BBB. Several studies have provided evidence of a novel and molecularly unknown drug/proton (H + )-antiporter that controls the permeation of many CNS-targeted drugs across the BBB (e.g. cocaine, clonidine, nicotine, oxycodone and diphenhydramine). Two studies have attempted to measure the function of this novel transporter by PET imaging. Auvity et al. used the radiolabeled histamine H1-receptor antagonist [ 11 C]diphenhydramine to image this transporter at the rat BBB [START_REF] Auvity | Diphenhydramine as a selective probe to study H+-antiporter function at the blood-brain barrier: Application to [11C]diphenhydramine positron emission tomography imaging[END_REF]). An evaluation of radiotracers for the H + -antiporter is complicated by a lack of suitable inhibitors for in vivo use. Auvity et al. therefore demonstrated with in situ brain perfusion that uptake transport of [ 14 C]diphenhydramine into rat brain can be inhibited by a large excess of unlabeled diphenhydramine (10 mM). In addition, they demonstrated poor brain penetration of radiolabeled metabolites, allowing for accurate quantification of CLuptake from blood into brain from early PET data. Gustafsson et al. performed combined PET imaging and microdialysis in rats with [ 11 C]oxycodone, which is also a substrate of the H + -antiporter [START_REF] Gustafsson | Combined PET and microdialysis for in vivo estimation of drug blood-brain barrier transport and brain unbound concentrations[END_REF]. However, the utility of this radiotracer for transporter imaging is most likely limited due to extensive metabolism and brain uptake of radiolabeled metabolites. To our knowledge, no attempts have so far been made to image OATP1A2 and OATP2B1 at the BBB, the latter being considered as target transporters for facilitating brain entry of therapeutic drugs [START_REF] Stieger | Drug transporters in the central nervous system[END_REF]. A potential complication in the development of PET tracers for these uptake transporters may be the fact that known transporter substrates are also recognized by efflux transporters at the BBB, which may mask the effect of the uptake transporters [START_REF] Taskar | Unmasking the Role of Uptake Transporters for Digoxin Uptake Across the Barriers of the Central Nervous System in Rat[END_REF][START_REF] Tournier | Effects of selected OATP and/or ABC transporter inhibitors on the brain and whole-body distribution of glyburide[END_REF].

Retina transporters

Drug distribution to the retina after systemic administration is controlled by the blood-ocular barriers, i.e. the blood-aqueous humor barrier and the blood-retinal barrier (BRB) [START_REF] Hosoya | Strategies for therapy of retinal diseases using systemic drug delivery: relevance of transporters at the blood-retinal barrier[END_REF]. The BRB is composed of retinal capillary endothelial cells (inner BRB) and retinal pigmental epithelial cells (outer BRB) [START_REF] Mannermaa | Drug transport in corneal epithelium and blood-retina barrier: emerging role of transporters in ocular pharmacokinetics[END_REF]. Similar to brain capillary endothelial cells, these cells are linked by tight junctions, which may limit paracellular diffusion of drugs. The cells comprising the BRB express SLC and ABC transporters, which may control the delivery of drugs from blood to the retina [START_REF] Hosoya | Strategies for therapy of retinal diseases using systemic drug delivery: relevance of transporters at the blood-retinal barrier[END_REF][START_REF] Mannermaa | Drug transport in corneal epithelium and blood-retina barrier: emerging role of transporters in ocular pharmacokinetics[END_REF][START_REF] Stieger | Drug transporters in the central nervous system[END_REF]. Pgp was shown to be localized at both the luminal membrane of retinal capillary endothelial cells and at the apical and basolateral membranes of retinal pigmental epithelial cells [START_REF] Hosoya | Strategies for therapy of retinal diseases using systemic drug delivery: relevance of transporters at the blood-retinal barrier[END_REF][START_REF] Mannermaa | Drug transport in corneal epithelium and blood-retina barrier: emerging role of transporters in ocular pharmacokinetics[END_REF].

Invasive experiments in rodents have revealed the functional impact of P-gp at this specific barrier [START_REF] Chapy | Blood-brain and retinal barriers show dissimilar ABC transporter impacts and concealed effect of P-glycoprotein on a novel verapamil influx carrier[END_REF]. Compared with the BBB, mBCRP-mediated efflux was shown to be less involved at the BRB, whereas mMRPs were involved to a similar degree at both barriers [START_REF] Chapy | Blood-brain and retinal barriers show dissimilar ABC transporter impacts and concealed effect of P-glycoprotein on a novel verapamil influx carrier[END_REF]. Using (R)-[ 11 C]verapamil PET imaging in healthy volunteers, Bauer et al. reported a significant increase in radiotracer distribution to the retina during P-gp inhibition, which provided first in vivo evidence for P-gp transport activity at the human BRB. Interestingly, the increase in retinal distribution was quantitatively less pronounced than for the brain (Bauer et al., 2017a), which correlated well with preclinical data obtained in rodents [START_REF] Chapy | Blood-brain and retinal barriers show dissimilar ABC transporter impacts and concealed effect of P-glycoprotein on a novel verapamil influx carrier[END_REF].

Lung transporters

Oral inhalation of therapeutic aerosols is used as a modality for local treatment of respiratory diseases (i.e. asthma and chronic obstructive pulmonary disease), but also as a route for systemic delivery of small molecule drugs and biologicals. The pharmacokinetic behavior of inhaled drugs is much more complicated than that of traditional routes of administration, with several sources of variability. There is accumulating evidence that membrane transporters belonging to the SLC and ABC families (e.g. organic cation / carnitine transporter 1 and 2 (OCTN1/2, SLC22A4/5), OCT1-3, MRP1, P-gp and BCRP) are expressed in bronchial, bronchiolar and alveolar epithelial cells forming the air-blood barrier [START_REF] Nickel | Transport mechanisms at the pulmonary mucosa: implications for drug delivery[END_REF][START_REF] Sakamoto | Quantitative expression of human drug transporter proteins in lung tissues: Analysis of regional, gender, and interindividual differences by liquid chromatography-tandem mass spectrometry[END_REF]. Drug transporters may be a source of variability in pulmonary PK of inhaled drugs and thus contribute to heterogeneity in treatment response and occurrence of systemic side effects [START_REF] Nickel | Transport mechanisms at the pulmonary mucosa: implications for drug delivery[END_REF]. PET and SPECT can be used to assess pulmonary disposition of inhaled radiopharmaceuticals [START_REF] Dolovich | Imaging drug delivery and drug responses in the lung[END_REF] and may thus also be of use to study the function of pulmonary transporters when radiolabeled transporter substrates are employed. So far, only very few studies have used SPECT or PET imaging to investigate pulmonary transporters. Okamura and colleagues reported that pulmonary elimination of radioactivity following intravenous injection of 6-bromo-7-[ 11 C]methylpurine is markedly reduced in Abcc1 (-/-) mice as compared with wild-type mice, suggesting that this radiotracer can be used to measure pulmonary mMRP1 function [START_REF] Okamura | Imaging of activity of multidrug resistance-associated protein 1 in the lungs[END_REF]. Two other studies used SPECT imaging to assess pulmonary disposition of inhaled [ 99m Tc]sestamibi, a substrate of P-gp and MRP1, in humans [START_REF] Perek | Involvement of the glutathione S-conjugate compounds and the MRP protein in Tc-99mtetrofosmin and Tc-99m-sestamibi uptake in glioma cell lines[END_REF][START_REF] Piwnica-Worms | Functional imaging of multidrug-resistant P-glycoprotein with an organotechnetium complex[END_REF]. Ruparelia et al. reported delayed pulmonary elimination of inhaled [ 99m Tc]sestamibi in smokers versus non-smokers and hypothesized that this was due to a smoke-induced upregulation of pulmonary P-gp [START_REF] Ruparelia | Pulmonary elimination rate of inhaled 99mTc-sestamibi radioaerosol is delayed in healthy cigarette smokers[END_REF]. Mohan et al. confirmed delayed [ 99m Tc]sestamibi elimination from the lungs of smokers versus non-smokers and more importantly found a correlation between [ 99m Tc]sestamibi elimination and a semi-quantitative measure of MRP1 expression in surgically resected lung tissue [START_REF] Mohan | Does the Clearance of Inhaled (99m)Tc-Sestamibi Correlate with Multidrug Resistance Protein 1 Expression in the Human Lung?[END_REF].

Placenta transporters

High expression levels of several ABC and SLC transporters have been reported in the placenta.

These transporters may play a role in controlling the distribution of drugs across the placental barrier [START_REF] Nishimura | Tissue-specific mRNA expression profiles of human ATPbinding cassette and solute carrier transporter superfamilies[END_REF], 2008). The use of nuclear imaging during pregnancy is restricted due to the lack of knowledge regarding the impact of ionizing radiation exposure on the developing fetus. However, PET imaging has been proposed to investigate drug exposure to the fetus in gestating non-human primates [START_REF] Benveniste | Maternal-fetal in vivo imaging: a combined PET and MRI study[END_REF]. Eyal and coworkers used [ 11 C]verapamil PET imaging in gestating macaques to study the role of P-gp at the placental barrier (Eyal et al., 2009a). Inhibition of P-gp using cyclosporine A dose-dependently increased [ 11 C]verapamil uptake by the fetal brain and liver [START_REF] Ke | Modeling cyclosporine A inhibition of the distribution of a P-glycoprotein PET ligand, 11C-verapamil, into the maternal brain and fetal liver of the pregnant nonhuman primate: impact of tissue blood flow and site of inhibition[END_REF]. These studies convincingly demonstrated the role of P-gp in limiting the placental permeation of its substrates in vivo.

Imaging transporter function in cancer tissues

Imaging techniques have provided convincing evidence for transporter-mediated multidrug resistance (MDR) in cancer tissues. The widespread availability of [ 99m Tc]sestamibi as a SPECT tracer in clinical routine allowed for its straightforward application in cancer patients to measure P-gp overexpression in tumors in vivo and predict MDR [START_REF] Derebek | 99mTc-MIBI scintigraphy in metastatic renal cell carcinoma: clinical validation of the relationship between 99mTc-MIBI uptake and P-glycoprotein expression in tumour tissue[END_REF]. In patients, P-gp inhibition resulted in a significant increase in the uptake [START_REF] Agrawal | Increased 99mTc-sestamibi accumulation in normal liver and drug-resistant tumors after the administration of the glycoprotein inhibitor, XR9576[END_REF][START_REF] Chen | Detection of in vivo P-glycoprotein inhibition by PSC 833 using Tc-99m sestamibi[END_REF] and, more importantly, a decrease in the wash-out rate of [ 99m Tc]sestamibi in tumors [START_REF] Taki | Assessment of Pglycoprotein in patients with malignant bone and soft-tissue tumors using technetium-99m-MIBI scintigraphy[END_REF], thus highlighting the functional impact of this transporter on the distribution of its substrates to cancer tissues. [ 99m Tc]tetrofosmin, another cationic cardiac imaging agent, has been evaluated as an alternative P-gp substrate to predict MDR in cancer [START_REF] Kao | Paclitaxel-Based chemotherapy for non-small cell lung cancer: predicting the response with 99mTctetrofosmin chest imaging[END_REF]. [ 99m Tc]sestamibi and [ 99m Tc]tetrofosmin are not transported by BCRP [START_REF] Tournier | Ibogaine labeling with 99mTc-tricarbonyl: synthesis and transport at the mouse blood-brain barrier[END_REF] but are both substrates of MRP1, which limits their utility as P-gp specific imaging agents in cancer [START_REF] Perek | Involvement of the glutathione S-conjugate compounds and the MRP protein in Tc-99mtetrofosmin and Tc-99m-sestamibi uptake in glioma cell lines[END_REF]. Pgp substrates radiolabeled with positron emitting isotopes have been further proposed to study MDR using PET [START_REF] Mann | Molecular Imaging of Membrane Transporters' Activity in Cancer: a Picture is Worth a Thousand Tubes[END_REF]. [ 11 C]Verapamil PET imaging has been performed in cancer patients, but failed to provide clinical utility [START_REF] Eary | Multiagent PET for risk characterization in sarcoma[END_REF][START_REF] Hendrikse | Dynamics of multidrug resistance: P-glycoprotein analyses with positron emission tomography[END_REF].

Radiolabeled analogs or derivatives of taxanes such as [ 11 C]docetaxel and [ 18 F]fluoropaclitaxel have been evaluated in cancer patients as predictors of successful therapy [START_REF] Kurdziel | Human dosimetry and preliminary tumor distribution of 18F-fluoropaclitaxel in healthy volunteers and newly diagnosed breast cancer patients using PET/CT[END_REF]van der Veldt et al., 2013avan der Veldt et al., , 2013b)).

Dysregulation of SLC transporters appears to be a common feature in many tumors [START_REF] El-Gebali | Solute carriers (SLCs) in cancer[END_REF]. In hepatocellular carcinoma, the expression of OATP1B3 decreases in parallel with tumor differentiation [START_REF] Kitao | The uptake transporter OATP8 expression decreases during multistep hepatocarcinogenesis: correlation with gadoxetic acid enhanced MR imaging[END_REF][START_REF] Miura | Distinct clinicopathological phenotype of hepatocellular carcinoma with ethoxybenzyl-magnetic resonance imaging hyperintensity: association with gene expression signature[END_REF][START_REF] Yoneda | Hypervascular hepatocellular carcinomas showing hyperintensity on hepatobiliary phase of gadoxetic acidenhanced magnetic resonance imaging: a possible subtype with mature hepatocyte nature[END_REF]. This decreased expression was found to be paralleled by a reduced gadoxetate-induced signal enhancement in the tumor [START_REF] Tsuboyama | Hepatocellular carcinoma: hepatocyte-selective enhancement at gadoxetic acid-enhanced MR imaging-correlation with expression of sinusoidal and canalicular transporters and bile accumulation[END_REF]. A correlation has been found between the expression of OATP1B1 and/or OATP1B3 in pathologic liver cells and signal enhancement [START_REF] Tsuboyama | Hepatocellular carcinoma: hepatocyte-selective enhancement at gadoxetic acid-enhanced MR imaging-correlation with expression of sinusoidal and canalicular transporters and bile accumulation[END_REF]. Focal nodular hyperplasia and hepatocellular adenoma are usually difficult to distinguish in the absence of contrast enhancement. This distinction is, however, highly important clinically [START_REF] Pastor | The role of organic anion transporters in diagnosing liver diseases by magnetic resonance imaging[END_REF]. Again, a correlation between OATP1B3 expression and intensity of the enhancement was found [START_REF] Doi | Focal nodular hyperplasia-like nodule with reduced expression of organic anion transporter 1B3 in alcoholic liver cirrhosis[END_REF][START_REF] Yoneda | Benign Hepatocellular Nodules: Hepatobiliary Phase of Gadoxetic Acid-enhanced MR Imaging Based on Molecular Background[END_REF]. The same tumors may, however, also express an efflux system for the contrast agent, e.g. MRP2 or MRP3 [START_REF] Kitao | Hepatocellular carcinoma: signal intensity at gadoxetic acid-enhanced MR Imaging--correlation with molecular transporters and histopathologic features[END_REF][START_REF] Thian | Liver-specific agents for contrast-enhanced MRI: role in oncological imaging[END_REF][START_REF] Tsuboyama | Hepatocellular carcinoma: hepatocyte-selective enhancement at gadoxetic acid-enhanced MR imaging-correlation with expression of sinusoidal and canalicular transporters and bile accumulation[END_REF][START_REF] Vilgrain | Insights into the diagnosis of hepatocellular carcinomas with hepatobiliary MRI[END_REF]. These examples highlight the limitations of determining in vivo transporter expression/function using imaging agents which are not specific to an individual transporter. An alternative approach to distinguish between these two lesions may be PET imaging with [ 18 F]fluorocholine. Three patients with hepatocellular adenoma were found to show reduced [ 18 F]fluorocholine uptake into the lesion, which correlated with a reduced expression of OCT1 and OCT3 [START_REF] Visentin | Impact of Organic Cation Transporters (OCT-SLC22A) on Differential Diagnosis of Intrahepatic Lesions[END_REF].

Discoveries and perspectives enabled by transporter imaging

Implications of the interplay between transporters and metabolism for imaging

As the liver is a central organ in drug metabolism and elimination, defining the transport systems involved in these processes as well as understanding the role of these transporters in the PK of drugs is a very active field of research [START_REF] Burt | Intraarterial Microdosing: A Novel Drug Development Approach, Proof-of-Concept PET Study in Rats[END_REF][START_REF] Hillyar | PET and SPECT Imaging for the Acceleration of Anti-Cancer Drug Development[END_REF][START_REF] Testa | PET Tracers To Study Clinically Relevant Hepatic Transporters[END_REF].

The interplay between transport and metabolism is termed metabolic channeling [START_REF] Srere | Enzyme-enzyme interactions and their metabolic role[END_REF]. The channeling of drugs via transporters into metabolism has been known of at the phenomenological level in clinics for quite some time and demonstrated by means of pharmacokinetic DDI studies [START_REF] Benet | The drug transporter-metabolism alliance: uncovering and defining the interplay[END_REF][START_REF] Custodio | Predicting drug disposition, absorption/elimination/transporter interplay and the role of food on drug absorption[END_REF]. One study tested the interaction of rifampicin with glyburide disposition in two settings: a single dose of rifampicin led to an increase of systemic glyburide exposure, while multiple dosing of rifampicin led to a considerable reduction of glyburide exposure [START_REF] Zheng | Elucidating rifampin's inducing and inhibiting effects on glyburide pharmacokinetics and blood glucose in healthy volunteers: unmasking the differential effects of enzyme induction and transporter inhibition for a drug and its primary metabolite[END_REF]. Tournier et al. mimicked this glyburide-rifampicin interaction using [ 11 C]glyburide PET imaging in non-human primates. Single dose rifampicin resulted in a 14-fold decrease in [ 11 C]glyburide uptake by the liver. Reduced liver uptake was associated with a pronounced 5-fold increase in [ 11 C]glyburide exposure in plasma. Interestingly, radiolabeled metabolites, which accounted for ~30% of total radioactivity in plasma, could not be detected in plasma after OATP inhibition [START_REF] Tournier | Effects of selected OATP and/or ABC transporter inhibitors on the brain and whole-body distribution of glyburide[END_REF] (Fig. 5). Imaging may thus be useful to investigate the interplay between transporter-mediated liver uptake and metabolic enzymes in controlling the liver metabolism of drugs. Metabolic channeling is gaining attention in the drug development and pharmacokinetic community [START_REF] Varma | Dealing with the complex drugdrug interactions: towards mechanistic models[END_REF][START_REF] Varma | Transporter-Enzyme Interplay: Deconvoluting Effects of Hepatic Transporters and Enzymes on Drug Disposition Using Static and Dynamic Mechanistic Models[END_REF]). An additional level of complexity is added by the fact that drug metabolites may also interact with (efflux) transporters, as exemplified for bosentan [START_REF] Fattinger | The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions[END_REF] and troglitazone [START_REF] Funk | Troglitazone-induced intrahepatic cholestasis by an interference with the hepatobiliary export of bile acids in male and female rats. Correlation with the gender difference in troglitazone sulfate formation and the inhibition of the canalicular bile salt export pump (Bsep) by troglitazone and troglitazone sulfate[END_REF]. The issues of metabolic flux and transporter-metabolism interplay have, to our knowledge, not been specifically addressed at the mechanistic level with imaging probes in different organs.

Imaging disease-induced changes in transporter expression at the BBB

Several PET studies have been conducted using racemic [ 11 C]verapamil or (R)-[ 11 C]verapamil to investigate cerebral P-gp function in different patient groups. However, due to the low sensitivity of [ 11 C]verapamil to measure moderate changes in P-gp expression/function at the BBB [START_REF] Wanek | Factors Governing P-Glycoprotein-Mediated Drug-Drug Interactions at the Blood-Brain Barrier Measured with Positron Emission Tomography[END_REF], most studies found only small differences between patients and control groups. A pilot PET study with (R)-[ 11 C]verapamil in seven patients with unilateral temporal lobe epilepsy failed to reveal significant differences in (R)-[ 11 C]verapamil distribution between epileptic brain tissue and contralateral healthy brain tissue [START_REF] Langer | Pharmacoresistance in epilepsy: a pilot PET study with the P-glycoprotein substrate R-[(11)C]verapamil[END_REF]. Van Assema et al. found no differences in (R)-[ 11 C]verapamil volume of distribution (VT) and K1 values, which were shown in previous studies to be the key parameters associated with P-gp function at the BBB [START_REF] Muzi | Imaging of cyclosporine inhibition of P-glycoprotein activity using 11C-verapamil in the brain: studies of healthy humans[END_REF][START_REF] Wagner | A pilot study to assess the efficacy of tariquidar to inhibit P-glycoprotein at the human blood-brain barrier with (R)-11C-verapamil and PET[END_REF]) between Alzheimer's disease (AD) patients and age-matched control subjects (van Assema et al., 2012a). On the other hand, the authors found a significantly higher non-displaceable binding potential (BPND) of (R)-[ 11 C]verapamil in AD patients, which they hypothesized to reflect a decrease in cerebral P-gp function (van Assema et al., 2012a). In a study by Deo et al., differences in brain distribution of [ 11 C]verapamil between AD patients and control subjects could only be detected when regional [ 11 C]verapamil K1 values were normalized to regional cerebral blood flow, which was lower in AD patients than in control subjects [START_REF] Deo | Activity of P-Glycoprotein, a β-Amyloid Transporter at the Blood-Brain Barrier, Is Compromised in Patients with Mild Alzheimer Disease[END_REF]. These data suggest that brain distribution of [ 11 C]verapamil depends on cerebral blood flow, as shown before for [ 11 C]N-desmethyl-loperamide [START_REF] Kreisl | Pglycoprotein function at the blood-brain barrier in humans can be quantified with the substrate radiotracer 11C-N-desmethyl-loperamide[END_REF][START_REF] Liow | Pglycoprotein function at the blood-brain barrier imaged using 11C-N-desmethyl-loperamide in monkeys[END_REF]. A series of studies used statistical parametrical mapping (SPM) analysis to investigate regional differences in brain distribution of racemic [ 11 C]verapamil in Parkinson's disease and depression and schizophrenia patients [START_REF] Bartels | Decreased blood-brain barrier P-glycoprotein function in the progression of Parkinson's disease, PSP and MSA[END_REF][START_REF] De Klerk | Locally increased P-glycoprotein function in major depression: a PET study with [11C]verapamil as a probe for P-glycoprotein function in the blood-brain barrier[END_REF][START_REF] De Klerk | Regional increase in P-glycoprotein function in the blood-brain barrier of patients with chronic schizophrenia: a PET study with [(11)C]verapamil as a probe for Pglycoprotein function[END_REF][START_REF] Kortekaas | Blood-brain barrier dysfunction in parkinsonian midbrain in vivo[END_REF]. These studies found small regional differences in [ 11 C]verapamil brain distribution, which were attributed to regional differences in P-gp function. There is evidence that P-gp expression at the BBB not only changes in the course of different neurological diseases, but also during healthy ageing. This could be of therapeutic relevance, as elderly people often take many different drugs which could show altered brain distribution if their brain distribution were limited by P-gp. A few studies used racemic [ 11 C]verapamil or (R)-[ 11 C]verapamil to compare cerebral P-gp function between elderly and young volunteers [START_REF] Bartels | Blood-brain barrier P-glycoprotein function decreases in specific brain regions with aging: a possible role in progressive neurodegeneration[END_REF][START_REF] Bauer | Age dependency of cerebral P-gp function measured with (R)-[11C]verapamil and PET[END_REF][START_REF] Toornvliet | Effect of age on functional P-glycoprotein in the blood-brain barrier measured by use of (R)-[(11)C]verapamil and positron emission tomography[END_REF]van Assema et al., 2012b). These studies consistently found small increases in radiotracer brain distribution, both on a global and regional level, in elderly versus young subjects, which pointed to an age-related, moderate decline in P-gp function at the BBB. A few studies employed a partial P-gp inhibition protocol, which showed improved sensitivity to measure moderate changes in P-gp expression/function at the BBB than baseline PET scans [START_REF] Bankstahl | A novel positron emission tomography imaging protocol identifies seizure-induced regional overactivity of P-glycoprotein at the blood-brain barrier[END_REF].

Feldmann et al. demonstrated that patients with pharmacoresistant unilateral temporal lobe epilepsy had a lower increase in (R)-[ 11 C]verapamil uptake in the ipsilateral hippocampus in response to tariquidar administration than healthy control subjects (24.5% vs. 65% increase in (R)-[ 11 C]verapamil K1) [START_REF] Feldmann | P-glycoprotein expression and function in patients with temporal lobe epilepsy: a casecontrol study[END_REF]. This attenuated response to tariquidar was consistent with increased P-gp expression in the hippocampus of pharmacoresistant epilepsy patients. Another study used cyclosporine A for partial P-gp inhibition at the BBB and found a significantly higher asymmetry in (R)-[ 11 C]verapamil brain concentrations between ipsilateral and contralateral temporal lobe brain regions of drug-resistant temporal lobe epilepsy patients as compared with drug-sensitive epilepsy patients and healthy control subjects [START_REF] Shin | Clinical Applications of Simultaneous PET/MR Imaging Using (R)-[11C]-Verapamil with Cyclosporin A: Preliminary Results on a Surrogate Marker of Drug-Resistant Epilepsy[END_REF].

Bauer et al. demonstrated that the partial P-gp inhibition protocol can also be used to visualize a reduction of P-gp at the BBB as occurs during healthy ageing (Bauer et al., 2017c). They found a significantly higher increase in (R)-[ 11 C]verapamil whole-brain VT in elderly versus young subjects (+40% vs. + 2%) in response to tariquidar administration. However, the partial P-gp inhibition protocol is difficult to use in patients due to safety concerns associated with P-gp inhibition and the concomitant use of medication. Clearly, to measure P-gp function at the BBB there is still a need for more sensitive PET tracers which do not require administration of a P-gp inhibitor and which lack brain-penetrant radiolabeled metabolites.

Imaging drug-drug interactions at the tissue level

Beside drug metabolizing enzymes, transporters may also be implicated in DDIs. In transportermediated DDIs, concomitant administration of two drugs which interact with the same transporters may lead to changes in the PK of the drugs as compared to when these drugs are dosed alone, due to mutual inhibition or saturation of ABC and SLC transporters controlling drug tissue distribution or excretion [START_REF] Giacomini | Membrane transporters in drug development[END_REF][START_REF] Lee | Evaluation of transporters in drug development: Current status and contemporary issues[END_REF]. Such DDIs can seriously affect drug safety and efficacy [START_REF] König | Transporters and Drug-Drug Interactions: Important Determinants of Drug Disposition and Effects[END_REF], and regulatory authorities therefore require assessment of the DDI risk of new drug candidates [START_REF]Guideline on the investigation of drug interactions[END_REF][START_REF] Fda | Clinical Drug Interaction Studies -Study Design, Data Analysis, and Clinical Implications -Guidance for Industry[END_REF]. In many cases, transporter-mediated DDIs lead to changes in drug plasma PK and can therefore be detected by monitoring plasma concentrations of drugs. In some cases, however, inhibition of transporters may predominantly affect drug tissue concentrations [START_REF] Kusuhara | In vitro-in vivo extrapolation of transporter-mediated clearance in the liver and kidney[END_REF][START_REF] Patilea-Vrana | Transport vs. Metabolism: What Determines the Pharmacokinetics and Pharmacodynamics of Drugs? Insights From the Extended Clearance Model[END_REF]. For instance, inhibition of efflux transporters in the canalicular membrane of hepatocytes or in the brush border membrane of kidney proximal tubule cells may lead to changes in liver and kidney concentrations of drugs. Nuclear imaging methods allow assessment of tissue concentrations of radiolabeled drugs and have therefore been proposed as a tool to investigate such silent DDIs [START_REF] Kusuhara | Imaging in the study of membrane transporters[END_REF][START_REF] Langer | Use of PET Imaging to Evaluate Transporter-Mediated Drug-Drug Interactions[END_REF][START_REF] Wulkersdorfer | Using positron emission tomography to study transporter-mediated drug-drug interactions in tissues[END_REF]. Consequently, several groups have developed radiolabeled probe substrates for measuring the activities of ABC and SLC transporters in different organs with PET (e.g. brain, liver and kidney) [START_REF] Langer | Use of PET Imaging to Evaluate Transporter-Mediated Drug-Drug Interactions[END_REF][START_REF] Marie | Imaging Probes and Modalities for the Study of Solute Carrier O (SLCO)-Transport Function In Vivo[END_REF][START_REF] Testa | PET Tracers To Study Clinically Relevant Hepatic Transporters[END_REF]. Most of these probes lack selectivity for a single ABC or SLC transporter.

Japanese researchers around Prof. Yuichi Sugiyama developed several PET probe substrates to measure the activities of sinusoidal uptake transporters in hepatocytes ((15R)-[ 11 C]TIC-Me, [START_REF] Kusuhara | Imaging in the study of membrane transporters[END_REF]. (15R)-[ 11 C]TIC-Me is rapidly converted into (15R)-[ 11 C]TIC in vivo. In one study it was shown that CLuptake,liver and CLint,bile of (15R)-[ 11 C]TIC-associated radioactivity were reduced in healthy human subjects after oral treatment with rifampicin, which was attributed to inhibition of sinusoidal OATPs (OATP1B1, OATP1B3) and possibly canalicular MRP2 [START_REF] Takashima | PET imaging-based evaluation of hepatobiliary transport in humans with (15R)-11C-TIC-Me[END_REF]. Similar results were obtained with [ 11 C]dehydropravastatin after rifampicin treatment in humans [START_REF] Kaneko | A clinical quantitative evaluation of hepatobiliary transport of [11C]Dehydropravastatin in humans using positron emission tomography[END_REF]. Metformin is a widely used oral antidiabetic drug. PET studies with [ 11 C]metformin revealed pronounced changes in liver and kidney distribution of [ 11 C]metformin in rodents when uptake transporters (mOCT1 and mOCT2) and efflux transporters (mMATE) were pharmacologically inhibited with cimetidine and pyrimethamine, respectively [START_REF] Shingaki | Quantitative Evaluation of mMate1 Function Based on Minimally Invasive Measurement of Tissue Concentration Using PET with [(11)C]Metformin in Mouse[END_REF]Jensen et al., 2016). The radiolabeled epidermal growth factor receptor-targeting tyrosine kinase inhibitor (TKI) [ 11 C]erlotinib was found to be transported in vitro at low concentrations as used for PET imaging by OATP2B1, but not by OATP1B1 and OATP1B3, whereas OATP2B1 transport was saturated at pharmacologic erlotinib concentrations. In vivo, liver uptake of [ 11 C]erlotinib was markedly lower after pre-treatment of healthy volunteers with a therapeutic erlotinib dose as compared with administration of a microdose of [ 11 C]erlotinib, which appeared to be caused by saturation of OATP2B1 transport (Bauer et al., 2017b) (Fig. 6). These data suggested that erlotinib may be a perpetrator of OATP2B1-mediated DDIs when combined with drugs which are mainly taken up into the liver by OATP2B1. Moreover, [ 11 C]erlotinib may be useful as an OATP2B1-specific probe substrate for PET studies.

[ 11 C]telmisartan, [ 11 C]dehydropravastatin)
The SPECT tracer [ 99m Tc]mebrofenin is a substrate of OATP1B1, OATP1B3, MRP2 and MRP3 (de Graaf et al., 2011;[START_REF] Ghibellini | Use of tc-99m mebrofenin as a clinical probe to assess altered hepatobiliary transport: integration of in vitro, pharmacokinetic modeling, and simulation studies[END_REF]. Pfeifer et al. demonstrated in a SPECT study in healthy volunteers that oral pretreatment with ritonavir decreased CLuptake,liver of [ 99m Tc]mebrofenin by 46%, which was attributed to inhibition of OATP1B1 and OATP1B3 [START_REF] Pfeifer | Effect of Ritonavir on (99m)Technetium-Mebrofenin Disposition in Humans: A Semi-PBPK Modeling and In Vitro Approach to Predict Transporter-Mediated DDIs[END_REF].

The entry of gadoxetate and BOPTA into hepatocytes is mediated by OATP1B1 and OATP1B3, and they are secreted into bile in unchanged form through MRP2. Kato et al. investigated the interaction between gadoxetate and 11 clinically used drugs in rats using MRI [START_REF] Kato | Gadolinium-ethoxybenzyl-diethylenetriamine-pentaacetic acid interaction with clinical drugs in rats[END_REF].

They found a significant decrease in contrast enhancement of the liver when rats were pre-treated with rifampicin, but concluded that a combination of gadoxetate and rifampicin is unlikely to occur in the clinic. One study in healthy volunteers investigated the interaction between gadoxetate and the macrolide antibiotic erythromycin, which inhibits OATP1B1 and OATP1B3 in vitro [START_REF] Seithel | The influence of macrolide antibiotics on the uptake of organic anions and drugs mediated by OATP1B1 and OATP1B3[END_REF], and failed to detect a significant effect of erythromycin on liver enhancement [START_REF] Huppertz | Evaluation of possible drug-drug interaction between gadoxetic acid and erythromycin as an inhibitor of organic anion transporting peptides (OATP)[END_REF].

PET imaging has been useful in evaluating the risk for DDIs at the level of ABC transporters expressed at the BBB, which may lead to increased brain distribution of drugs and CNS side effects [START_REF] Eyal | Drug interactions at the blood-brain barrier: fact or fantasy[END_REF]. In a seminal position paper, Kalvass and colleagues argued that transporter-mediated DDIs at the BBB are unlikely to occur in clinical practice as most marketed drugs do not achieve high enough unbound plasma concentrations to lead to significant efflux transporter inhibition at the BBB [START_REF] Kalvass | Why clinical modulation of efflux transport at the human blood-brain barrier is unlikely: the ITC evidence-based position[END_REF]. Exceptions are the immunosuppressant drug cyclosporine A, which was shown to increase brain exposure of [ 11 C]verapamil 1.8-fold when administered as an intravenous infusion concurrent with the PET scan [START_REF] Sasongko | Imaging P-glycoprotein transport activity at the human blood-brain barrier with positron emission tomography[END_REF].

The antiarrhythmic agent quinidine was also shown to exert a small P-gp inhibitory effect at the human BBB at clinically relevant plasma concentrations [START_REF] Liu | Modulation of P-glycoprotein at the Human Blood-Brain Barrier by Quinidine or Rifampin Treatment: A Positron Emission Tomography Imaging Study[END_REF]. In contrast to the majority of marketed drugs, several non-marketed, experimental P-gp inhibitors which were originally developed as MDR reversal agents (e.g. elacridar, tariquidar and valspodar) [START_REF] Szakács | Targeting multidrug resistance in cancer[END_REF] were shown to lead to significant increases in brain distribution of P-gp substrates.

For instance, up to 4-to 5-fold increases in brain distribution of (R)-[ 11 C]verapamil or [ 11 C]Ndesmethyl-loperamide were observed in healthy volunteers when tariquidar was intravenously infused during the PET scan [START_REF] Bauer | Approaching complete inhibition of P-glycoprotein at the human blood-brain barrier: an (R)-[11C]verapamil PET study[END_REF][START_REF] Kreisl | Increased permeability-glycoprotein inhibition at the human blood-brain barrier can be safely achieved by performing PET during peak plasma concentrations of tariquidar[END_REF]. These studies do not represent realistic scenarios for clinically relevant transporter-mediated DDIs at the BBB as they employed non-marketed drugs dosed intravenously at rather high doses. Nevertheless, these data suggest that efflux transporter inhibition at the human BBB is in principle feasible and may be exploited to enhance brain distribution of drugs targeted to the brain.

Imaging the impact of transporter gene polymorphisms on transporter function

Polymorphisms in the genes encoding transport proteins were shown to be associated with alterations in transporter activity and expression, leading to inter-individual variability in drug disposition and drug response [START_REF] Ieiri | Functional significance of genetic polymorphisms in P-glycoprotein (MDR1, ABCB1) and breast cancer resistance protein (BCRP, ABCG2)[END_REF][START_REF] König | Pharmacogenomics of human OATP transporters[END_REF][START_REF] Maeda | Impact of genetic polymorphisms of transporters on the pharmacokinetic, pharmacodynamic and toxicological properties of anionic drugs[END_REF][START_REF] Niemi | Organic anion transporting polypeptide 1B1: a genetically polymorphic transporter of major importance for hepatic drug uptake[END_REF]. Imaging with radiolabeled transporter substrates is an obvious way to assess the functional impact of these genetic variants in different tissues, and a handful of clinical studies have so far been published.

Two studies assessed the impact of a combination of three different ABCB1 SNPs on brain distribution of racemic [ 11 C]verapamil [START_REF] Brunner | Influence of functional haplotypes in the drug transporter gene ABCB1 on central nervous system drug distribution in humans[END_REF][START_REF] Takano | Evaluation of in vivo P-glycoprotein function at the blood-brain barrier among MDR1 gene polymorphisms by using 11C-verapamil[END_REF]. Both studies failed to detect differences in [ 11 C]verapamil brain distribution between healthy homozygous carriers of the TTT haplotype (c.3435T, c.2677T, and c.1236T) and homozygous carriers (controls) of the CGC haplotype (c.3435C, c.2677G, and c.1236C). Interestingly, a study in AD patients by van Assema found an association between T dose in the c.C1236T, c.G2677T and c.C3435T SNPs and BPND of (R)-[ 11 C]verapamil (van Assema et al., 2012c). Carriers of the variant alleles had a higher BPND than non-carriers, which was interpreted by the authors as decreased P-gp function at the BBB. The same study found no effect of these ABCB1 SNPs on brain distribution of (R)-[ 11 C]verapamil in healthy control subjects, which confirmed the results obtained by Brunner and Takano [START_REF] Brunner | Influence of functional haplotypes in the drug transporter gene ABCB1 on central nervous system drug distribution in humans[END_REF][START_REF] Takano | Evaluation of in vivo P-glycoprotein function at the blood-brain barrier among MDR1 gene polymorphisms by using 11C-verapamil[END_REF] and suggested that genetic variations in the ABCB1 gene might affect P-gp function or expression at the BBB only when Pgp function is already compromised. The most important SNP in the ABCG2 gene is c.421C>A. This non-synonymous SNP has been associated with diminished expression of BCRP in various tissues, such as the liver, placenta and intestine [START_REF] Kobayashi | Functional assessment of ABCG2 (BCRP) gene polymorphisms to protein expression in human placenta[END_REF][START_REF] Prasad | Interindividual variability in the hepatic expression of the human breast cancer resistance protein (BCRP/ABCG2): effect of age, sex, and genotype[END_REF] and with changes in plasma PK of BCRP substrate drugs, such as sulfasalazine and rosuvastatin [START_REF] Keskitalo | ABCG2 polymorphism markedly affects the pharmacokinetics of atorvastatin and rosuvastatin[END_REF][START_REF] Yamasaki | Pharmacogenetic characterization of sulfasalazine disposition based on NAT2 and ABCG2 (BCRP) gene polymorphisms in humans[END_REF]. Bauer et al. used a newly developed PET protocol [START_REF] Wanek | A novel PET protocol for visualization of breast cancer resistance protein function at the bloodbrain barrier[END_REF] to measure the impact of this ABCG2 SNP on the function of BCRP at the BBB [START_REF] Bauer | Pilot PET Study to Assess the Functional Interplay Between ABCB1 and ABCG2 at the Human Blood-Brain Barrier[END_REF]. This PET protocol is based on PET scans with the dual P-gp/BCRP substrate [ 11 C]tariquidar, in which unlabeled tariquidar is co-administered to inhibit P-gp and eliminate the contribution of P-gp to brain distribution of the dual substrate. Bauer reported 1.7fold higher [ 11 C]tariquidar VT values in heterozygous SNP carriers (c.421CA) as compared with non-carriers (c.421CC), which was consistent with reduced BCRP transport activity at the BBB of SNP carriers [START_REF] Bauer | Pilot PET Study to Assess the Functional Interplay Between ABCB1 and ABCG2 at the Human Blood-Brain Barrier[END_REF]. Interestingly, no difference in [ 11 C]tariquidar brain distribution was observed between carriers and non-carriers in PET scans without tariquidar coadministration, which indicated that the effect of the ABCG2 SNP was masked in presence of functional P-gp. Variants in the SLC22A1 gene, which encodes OCT1, were shown to affect OCT1 function and expression and have been associated with altered response to the oral antidiabetic drug metformin compared with carriers of the reference allele, without affecting metformin systemic exposure [START_REF] Zamek-Gliszczynski | Emerging Clinical Importance of Hepatic Organic Cation Transporter 1 (OCT1) in Drug Pharmacokinetics, Dynamics, Pharmacogenetic Variability, and Drug Interactions[END_REF]. This is because the liver is the target organ of metformin distribution and pharmacologic activity, but the drug is ultimately eliminated by the kidney. Sundelin et al. showed that exposure of [ 11 C]metformin in the liver was significantly lower in carriers of p.M420del and p.R61C variants in SLC22A1 after both oral and intravenous administration of the PET tracer [START_REF] Sundelin | Genetic Polymorphisms in Organic Cation Transporter 1 Attenuates Hepatic Metformin Exposure in Humans[END_REF]. This supported the notion that genetic SLC22A1 variants may affect metformin response. Two studies found diminished liver enhancement with gadoxetate in carriers of SLCO1B1 SNPs without changes in plasma PK of gadoxetate, suggesting that genetic transporter variants may be signal confounders in gadoxetate-enhanced diagnostic liver MRI [START_REF] Nassif | Visualization of hepatic uptake transporter function in healthy subjects by using gadoxetic acid-enhanced MR imaging[END_REF][START_REF] Okubo | Polymorphisms in the organic anion transporting polypeptide genes influence liver parenchymal enhancement in gadoxetic acid-enhanced MRI[END_REF]. One study examined liver distribution of [ 99m Tc]mebrofenin in healthy volunteers and patients with nonalcoholic steatohepatitis and found a diminished CLuptake,liver of [ 99m Tc]mebrofenin in healthy volunteers who were carriers of low/intermediate function SLCO1B1 genetic variants [START_REF] Ali | Transporter-Mediated Alterations in Patients With NASH Increase Systemic and Hepatic Exposure to an OATP and MRP2 Substrate[END_REF]. It can be expected that transporter polymorphisms will also affect tissue uptake of other clinically used diagnostic radiotracers or contrast agents and should therefore be considered in the interpretation of imaging data.

Animal to clinical extrapolation

Species differences have been reported in the substrate recognition and transport efficacy of ABC and SLC transporters [START_REF] Kim | Relationship between drugs and functional activity of various mammalian P-glycoproteins (ABCB1)[END_REF][START_REF] Li | Identification of interspecies difference in efflux transporters of hepatocytes from dog, rat, monkey and human[END_REF][START_REF] Xia | Comparison of species differences of P-glycoproteins in beagle dog, rhesus monkey, and human using Atpase activity assays[END_REF][START_REF] Yamazaki | In vitro substrate identification studies for p-glycoprotein-mediated transport: species difference and predictability of in vivo results[END_REF].

For instance, it has been shown that antiseizure drugs are good substrates of mouse P-gp (mABCB1A), whereas they are only weakly transported by human P-gp [START_REF] Baltes | Differences in the transport of the antiepileptic drugs phenytoin, levetiracetam and carbamazepine by human and mouse P-glycoprotein[END_REF][START_REF] Luna-Tortós | Several major antiepileptic drugs are substrates for human P-glycoprotein[END_REF]. The PET tracer

[ 18 F]MPPF (4-(2'-methoxyphenyl)-1-[2'-(N-2"-pyridinyl)-p- [ 18 F]
fluorobenzamido]ethylpiperazine) was shown to be transported by mABCB1A, but not by human P-gp [START_REF] Tournier | Transport of selected PET radiotracers by human P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2): an in vitro screening[END_REF][START_REF] Tournier | Discrepancies in the P-glycoprotein-mediated transport of (18)F-MPPF: a pharmacokinetic study in mice and non-human primates[END_REF]. Direct murine orthologues do not exist for some drug transporters. For example, in the sinusoidal membrane of human hepatocytes the SLCO transporters OATP1B1, OATP1B3 and OATP2B1 are expressed, whereas in mouse hepatocytes mOATP1B2, mOATP2B1, mOATP1A1 and mOATP1A4 are expressed [START_REF] Durmus | The impact of Organic Anion-Transporting Polypeptides (OATPs) on disposition and toxicity of antitumor drugs: Insights from knockout and humanized mice[END_REF]. mOATP1B2 is the mouse orthologue of human OATP1B1 and OATP1B3. mOATP1A1 and mOATP1A4 are mouse orthologues of human OATP1A2, which in the human liver is not expressed in the sinusoidal hepatocyte membrane but in epithelial cells of the bile duct. Owing to these differences, preclinical data on OATP-mediated transport in rodent liver is notoriously difficult to extrapolate to humans [START_REF] Durmus | The impact of Organic Anion-Transporting Polypeptides (OATPs) on disposition and toxicity of antitumor drugs: Insights from knockout and humanized mice[END_REF] (Fig. 6). The advent of quantitative targeted absolute proteomics has provided a tool to quantify absolute expression levels of transport proteins in different organs and tissues [START_REF] Ohtsuki | Quantitative targeted absolute proteomics-based ADME research as a new path to drug discovery and development: methodology, advantages, strategy, and prospects[END_REF]. These studies, too, revealed pronounced species differences in transporter expression. For instance, while BCRP was shown to be the most abundant ABC transporter at the human BBB, P-gp (mABCB1A) was found to be more abundant at the mouse BBB than mBCRP [START_REF] Shawahna | Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels[END_REF][START_REF] Uchida | Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors[END_REF].

While mMRP4 was detected as the third most abundant ABC transporter at the mouse BBB, it has not yet been detected at the human BBB [START_REF] Uchida | Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors[END_REF]. Such species differences are expected to lead to species differences in tissue distribution and excretion of PET/SPECT radiotracers and MRI contrast agents which are transporter substrates. Owing to the availability of dedicated small-animal PET, SPECT and MRI systems, differences between rodents and humans can be directly investigated in a translational approach using the same technology.

However, several caveats have to be kept in mind. One important issue is the fact that imaging studies in rodents are usually performed under anaesthesia (e.g. isoflurane), while this is not the case in humans. Second, it is well known that species differences also exist in metabolism pathways of xenobiotics [START_REF] Martignoni | Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition and induction[END_REF]. Therefore, species differences in radiotracer metabolism may mask species differences in drug transporters. Third, several key physiological parameters differ between rodents and humans (e.g. organ blood flow) [START_REF] Davies | Physiological parameters in laboratory animals and humans[END_REF], which may also exert an influence on tissue distribution of radiotracers. Some imaging studies have attempted to address these issues specifically. Syvänen et al. assessed differences between rats, monkeys and humans in brain distribution of three radiolabeled P-gp substrates ([ 11 C]verapamil, [ 11 C]GR205171 and [ 18 F]altanserin). These authors found higher brain-to-plasma ratios of radioactivity in monkeys or humans as compared with rats, and higher increases in brain distribution of these radiotracers following cyclosporine A treatment in rats than in monkeys [START_REF] Syvänen | Species differences in blood-brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport[END_REF]. Similarly, P-gp inhibition with tariquidar at comparable tariquidar plasma concentrations produced a higher increase in brain uptake of (R)-[ 11 C]verapamil in rats than in humans [START_REF] Kuntner | Dose-response assessment of tariquidar and elacridar and regional quantification of Pglycoprotein inhibition at the rat blood-brain barrier using (R)-[(11)C]verapamil PET[END_REF][START_REF] Bauer | Pgpmediated interaction between (R)-[11C]verapamil and tariquidar at the human blood-brain barrier: a comparison with rat data[END_REF][START_REF] Wagner | A pilot study to assess the efficacy of tariquidar to inhibit P-glycoprotein at the human blood-brain barrier with (R)-11C-verapamil and PET[END_REF] (Fig. 4). In accordance with this, in vivo half-maximum inhibitory concentrations for enhancement of brain uptake of (R)-[ 11 C]verapamil by tariquidar were determined to be lower in rats (873 nM) than in humans (2,248 nM) [START_REF] Bauer | Approaching complete inhibition of P-glycoprotein at the human blood-brain barrier: an (R)-[11C]verapamil PET study[END_REF][START_REF] Kuntner | Dose-response assessment of tariquidar and elacridar and regional quantification of Pglycoprotein inhibition at the rat blood-brain barrier using (R)-[(11)C]verapamil PET[END_REF] (Fig. 4). This stands in contrast with data for cyclosporine A, for which increases in brain uptake of [ 3 H]verapamil achieved in rats were found to predict increases in brain uptake of [ 11 C]verapamil in humans [START_REF] Hsiao | Verapamil P-glycoprotein transport across the rat blood-brain barrier: cyclosporine, a concentration inhibition analysis, and comparison with human data[END_REF]. Wanek et al. systematically assessed possible reasons for species differences in brain distribution of (R)-[ 11 C]verapamil and [ 11 C]N-desmethyl-loperamide [START_REF] Wanek | Factors Governing P-Glycoprotein-Mediated Drug-Drug Interactions at the Blood-Brain Barrier Measured with Positron Emission Tomography[END_REF]. They concluded that species differences in metabolism along with brain uptake of radiolabeled metabolites precluded an assessment of species differences in P-gp transport activity at the BBB with these two radiotracers. Consequently, for an assessment of species differences in ABC and SLC transporter activities, the availability of radiotracers that are not metabolized over the duration of a PET scan in all investigated species is mandatory.

No general recommendations can be made as to which preclinical species better predicts the pharmacokinetic disposition of an imaging probe in humans. For instance, with respect to Pgp/BCRP activity at the BBB, non-human primates have been identified as a more appropriate model of the human BBB than rodents [START_REF] Ito | Quantitative membrane protein expression at the blood-brain barrier of adult and younger cynomolgus monkeys[END_REF][START_REF] Uchida | Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors[END_REF]. In recent years, efforts have been directed toward the development of mouse models humanized for certain ABC or SLC transporters or metabolizing enzymes [START_REF] Choo | Leveraging Humanized Animal Models to Understand Human Drug Disposition: Opportunities, Challenges, and Future Directions[END_REF]. These mouse models still suffer from certain limitations. In most cases, for instance, the models are only humanized for a single transporter gene and compensatory up-/down-regulation of other transporters or enzymes may occur. Nevertheless, these animal models may ultimately allow a better translation of transporter imaging results from mice to humans. Finally, it should be emphasized that PET and SPECT use sub-pharmacological doses (microdoses) of imaging probes, so that toxicity issues are not usually of concern in imaging experiments in humans. In many cases, transporter imaging probes are derived from known drugs or drug metabolites, and regulatory hurdles preventing rapid translation of PET tracers from preclinical species into humans are much smaller than in conventional drug development (ICH Expert Working Group, 2009). Consequently, while rodent experiments may be very useful in developing and improving methodological aspects and addressing certain questions, such as the tissue content of radiolabeled metabolites, small pilot studies in healthy human volunteers may be the best way to assess the suitability of a novel transporter imaging probe for clinical use.

Pharmacokinetic imaging to study the fate of drugs at the site of action

The study of drug transporters with imaging modalities most commonly relies on the use of prototypical probe substrates. These are ideally designed to be specific to one transporter with high transport capacity, in order to generate substantial "contrast" between conditions where transporter activity is absent or present [START_REF] Kannan | Imaging the function of P-glycoprotein with radiotracers: pharmacokinetics and in vivo applications[END_REF][START_REF] Wanek | Factors Governing P-Glycoprotein-Mediated Drug-Drug Interactions at the Blood-Brain Barrier Measured with Positron Emission Tomography[END_REF]. However, quantitative data obtained with one prototypical probe substrate for one transporter might not be readily translated to other substrates of this transporter. Indeed, xenobiotics have different affinities for transporters, and scaling factors and suitable models have to be considered to extrapolate the in vivo impact of transporters on the tissue permeation of drugs of interest [START_REF] Kalvass | Why clinical modulation of efflux transport at the human blood-brain barrier is unlikely: the ITC evidence-based position[END_REF][START_REF] Matsuda | A Prediction Method for P-glycoprotein-Mediated Drug-Drug Interactions at the Human Blood-Brain Barrier From Blood Concentration-Time Profiles, Validated With PET Data[END_REF]. Moreover, most drugs are often substrates of multiple transporters and considerable overlap between ABC and SLC substrates is commonly observed [START_REF] Gui | Development of a cell-based highthroughput assay to screen for inhibitors of organic anion transporting polypeptides 1B1 and 1B3[END_REF][START_REF] Matsson | Identification of novel specific and general inhibitors of the three major human ATP-binding cassette transporters P-gp, BCRP and MRP2 among registered drugs[END_REF]. Substrates of influx transporters may also undergo efflux transport at the same interface. In this situation, the overall net flux of the drug results from the competition between the influx and efflux component, which is difficult to predict in the real-life situation [START_REF] Chapy | Blood-brain and retinal barriers show dissimilar ABC transporter impacts and concealed effect of P-glycoprotein on a novel verapamil influx carrier[END_REF][START_REF] Taskar | Unmasking the Role of Uptake Transporters for Digoxin Uptake Across the Barriers of the Central Nervous System in Rat[END_REF].

As an alternative to the use of prototypical probe substrates, direct isotopic labeling of the drug of interest may be employed. Theoretically, the study of drug distribution in selected organs and exposure to target/vulnerable tissues is feasible. However, careful interpretation of PET data is required to avoid any misinterpretation regarding drug exposure to investigated tissues. In most situations, radioactivity in tissues cannot be straightforwardly converted into local drug concentrations at the sites of action [START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF]. Conventional pharmacokinetic studies accurately measure the free ligand in plasma and are able to detect the presence of metabolites using analytical chemistry methods. However, PET-based kinetic imaging does not distinguish between radioactivity associated with the vascular content, the free, bound, and parent compounds, and radiolabeled metabolites in tissues [START_REF] Pike | PET radiotracers: crossing the blood-brain barrier and surviving metabolism[END_REF].

So far, pharmacologic imaging has predominantly focused on the interaction of drugs with their respective molecular targets in tissue through receptor occupancy (now named target engagement) studies [START_REF] Willmann | Molecular imaging in drug development[END_REF]. Advances in PET data acquisition and modeling have considerably enhanced the applicability of imaging biodistribution studies and paved the way for what could be named pharmacokinetic imaging. When drugs are amenable to radiolabeling, it is now possible to investigate the interplay of membrane transporters in controlling the clearance and tissue distribution of drugs. Using this approach, the impact of SLC and ABC transporters has been revealed in a dynamic fashion for glyburide [START_REF] Tournier | Effects of selected OATP and/or ABC transporter inhibitors on the brain and whole-body distribution of glyburide[END_REF], metformin (Jensen et al., 2016), sulpiride [START_REF] Takano | Possible Role of Organic Cation Transporters in the Distribution of [11C]Sulpiride, a Dopamine D2Receptor Antagonist[END_REF], metoclopramide [START_REF] Pottier | Imaging the Impact of the P-Glycoprotein (ABCB1) Function on the Brain Kinetics of Metoclopramide[END_REF], ciprofloxacin [START_REF] Wanek | Influence of 24-Nor-Ursodeoxycholic Acid on Hepatic Disposition of [(18)F]Ciprofloxacin, a Positron Emission Tomography Study in Mice[END_REF] and erlotinib (Bauer et al., 2017b;[START_REF] Traxl | Breast Cancer Resistance Protein and P-Glycoprotein Influence In Vivo Disposition of 11C-Erlotinib[END_REF], to cite a few examples. This emerging approach is increasingly regarded as a means to elucidate the overall impact of membrane transporters on drug distribution to tissues in animals and humans [START_REF] Giacomini | Membrane transporters in drug development[END_REF]. A step forward in modern pharmacology will be to investigate transporter-mediated drug uptake by tissues as a mediator of local drug exposure to tissues, and ultimately to assess or predict the contribution of membrane transporters to pharmacodynamics and toxicity. This approach is gaining attention for the study of drug delivery to sanctuary tissues, mainly in cancer.

For instance, the poor CNS permeation of TKIs is now accepted as a determinant of the poor response of CNS malignancies, including brain metastasis, to this class of molecularly targeted therapy [START_REF] Holohan | Cancer drug resistance: an evolving paradigm[END_REF][START_REF] Camidge | Acquired resistance to TKIs in solid tumours: learning from lung cancer[END_REF]. PET using radiolabeled analogs of TKIs can be regarded as a useful method to compare their CNS penetration in humans [START_REF] Verheijen | Molecular Imaging of ABCB1/ABCG2 Inhibition at the Human Blood Brain Barrier using Elacridar and 11C-Erlotinib PET[END_REF] and non-human primates, a relevant animal model of the human BBB [START_REF] Ballard | Preclinical Comparison of Osimertinib with Other EGFR-TKIs in EGFR-Mutant NSCLC Brain Metastases Models, and Early Evidence of Clinical Brain Metastases Activity[END_REF]Collier et al., 2017aCollier et al., , 2017b)). Virtually all members of the clinically important class of TKIs were identified as dual P-gp/BCRP substrates, which provides a mechanistic explanation for their low brain penetration [START_REF] Agarwal | Breast cancer resistance protein and P-glycoprotein in brain cancer: two gatekeepers team up[END_REF][START_REF] Durmus | Apical ABC transporters and cancer chemotherapeutic drug disposition[END_REF]. Recent studies used [ 11 C]erlotinib PET imaging to evaluate strategies to improve the brain delivery of erlotinib. P-gp/BCRP-mediated efflux of erlotinib at the BBB was inhibited using an intravenous infusion of high-dose elacridar in non-human primates [START_REF] Tournier | Strategies to Inhibit ABCB1-and ABCG2-Mediated Efflux Transport of Erlotinib at the Blood-Brain Barrier: A PET Study on Nonhuman Primates[END_REF]. However, P-gp/BCRP inhibition could not be achieved using an oral formulation of elacridar in humans, most likely due to the low oral bioavailability of this inhibitor [START_REF] Verheijen | Molecular Imaging of ABCB1/ABCG2 Inhibition at the Human Blood Brain Barrier using Elacridar and 11C-Erlotinib PET[END_REF].

Despite its methodological limitations, pharmacokinetic imaging is a potentially powerful approach to evaluating transporter-based strategies to improve the local delivery and/or tissue selectivity of drugs at their sites of action. Applications may not only be restricted to research in cancer therapy, but may also be extended to neurological or infectious diseases for which sanctuary sites are suspected to account for poor or incomplete therapeutic response in patients [START_REF] Rizk | Importance of Drug Pharmacokinetics at the Site of Action[END_REF]. Moreover, radiolabeled drugs may find use in evaluating protective pharmacologic strategies to mitigate organ toxicities of certain drugs (e.g. nephrotoxicity) [START_REF] Manohar | Cisplatin nephrotoxicity: a review of the literature[END_REF][START_REF] Reichart | Renal Protection Strategies after Heart Transplantation[END_REF].

Expert opinion and perspectives

Imaging methods are increasingly regarded as innovative means to explore drug transporters in vivo. Advances in the development of imaging probes dedicated to the study of drug transporter function has provided new insights into the role of drug transporters at several blood-tissue interfaces. However, imaging techniques still suffer from several technical and methodological limitations, which restrict their applicability to transporter studies. Limitations related to the inability of nuclear imaging methods to distinguish parent compound from radiolabeled metabolites have been discussed in this review article. Therefore, metabolically stable probe substrates are clearly preferred for transporter imaging studies. Regulatory authorities request that pharmaceutical companies examine the interaction of new drug candidates and their metabolites with ABC and SLC transporters to assess the risk for the occurrence of DDIs [START_REF]Guideline on the investigation of drug interactions[END_REF][START_REF] Fda | Clinical Drug Interaction Studies -Study Design, Data Analysis, and Clinical Implications -Guidance for Industry[END_REF]. Hence, it can be assumed that a wealth of unpublished data are available on new chemical entities which never reached the market but which may prove suitable for the development of transporter imaging probes. Clearly, the field may benefit considerably from public-private partnerships between academic researchers and pharmaceutical companies in order to advance transporter imaging. A major challenge in the imaging of transporters is related to the broadly overlapping substrate specificities of most ABC and SLC transporters, so that true transporter selectivity may be difficult to achieve in practice. However, the introduction of carefully matched, clinically feasible combinations of radiolabeled probe substrates with prototypical transporter inhibitors may prove beneficial in revealing the role played by specific ABC and SLC transporters in certain organ systems. For instance, lack of selectivity of a radiolabeled probe substrate for a given ABC or SLC transporter may be mitigated by combining it with a transporter inhibitor which inhibits only one of the ABC and SLC transporters by which the probe substrate is transported. Therefore, the screening of currently available, clinically applicable inhibitors with respect to their transporter selectivity profiles should be encouraged in order to yield effective substrate-inhibitor pairs for transporter imaging. In this framework, imaging of drug transporters may also clearly benefit from medicinal chemistry and structure-activity relationship studies aimed at improving the selectivity of transporter substrates and inhibitors [START_REF] Ekins | Computational Modeling to Accelerate the Identification of Substrates and Inhibitors For Transporters That Affect Drug Disposition[END_REF]. It should also be noted that probe substrates which lack transporter selectivity in one organ may still prove suitable to selectively study a certain transporter in another organ. Therefore, available radiotracers should be further evaluated in organs and at blood-tissue interfaces which have not yet been the major focus of transporter studies (e.g. BRB, placenta and lungs). Another limitation in the development of transporter imaging probes is related to difficulties in extrapolating preclinical results to humans.

In other words, imaging probes which were identified as suitable for transporter imaging in rodents may fail to perform in humans. This is a general problem which is also encountered in drug development. This risk may be mitigated by the introduction of more refined and better standardized preclinical assessment approaches, such as humanized mouse models, or more advanced cell culture experiments, such as sandwich-cultured hepatocytes. However, the key strength of imaging methods is that owing to their non-invasive or minimally invasive nature and the administration of very low, sub-pharmacological doses of the imaging probes, they are highly translational. Given the existence of sometimes pronounced species differences in transporters or metabolizing enzymes, and given the current availability of several prototypical transporter inhibitors for clinical use, the best way to characterize the clinical suitability of new imaging probes may ultimately be the performance of small pilot studies in healthy human volunteers. This approach may also be very useful in the assessment of transporter-mediated DDIs of new drug candidates in early clinical drug development. However, further studies with radiolabeled probe substrates based on marketed model drugs and prototypical inhibitors are needed to refine currently available analytical methodology, such as pharmacokinetic modeling approaches, taking into account the inherent limitations of imaging approaches, and to highlight the power of this approach in drug development. Another limitation of imaging tools is their high costs and the requirement for specialized research infrastructure, in particular for PET imaging. This is where less cost-intensive imaging approaches which are better suited to a hospital environment come into play. The discovery that MRI contrast agents or SPECT tracers interact with ABC and SLC transporters was mostly serendipitous, and so far hardly any systematic efforts have been made The exchange of 11 C-CSar between the compartments is described by fitted parameters: K 1 (mL blood/min/mL liver tissue), k2 (min -1 ), k3 (min -1 ), and k5 (min -1 ). The amount of 11 C-CSar in the blood compartment at time t is Cin(t) multiplied by Vblood (the fraction of blood in the liver tissue, mL blood/mL liver tissue). Cin(t) is the flow-weighted mixed input of 11 C-CSar to the liver from the hepatic artery and the portal vein, which was calculated from the arterial blood concentrations measured by arterial blood sampling. The amount of 11 C-CSar in the hepatocyte compartment at time t is Chep(t) (concentration of 11 C-CSar in hepatocytes, kBq/mL hepatocytes) multiplied by Vhep (the fraction of hepatocytes in the liver tissue, mL hepatocytes/mL liver tissue). The amount of 11 C-CSar at time t in the intrahepatic bile ducts is Cbile(t) (concentration of 11 C-CSar in intrahepatic bile ducts; kBq/mL bile) multiplied by Vbile (the fraction of intrahepatic bile ducts in the liver tissue, mL bile ducts/mL liver tissue) (reproduced with permission from (Ørntoft et al., 2017)). Concentration-response curves in rats (c) and humans (d) for enhancement of brain uptake of (R)-[ 11 C]verapamil (expressed as volume of distribution, V T ). Half-maximum inhibitory concentrations (IC50) of tariquidar were lower in rats than in humans. Adapted from [START_REF] Bauer | Pgpmediated interaction between (R)-[11C]verapamil and tariquidar at the human blood-brain barrier: a comparison with rat data[END_REF][START_REF] Kell | What would be the observable consequences if phospholipid bilayer diffusion of drugs into cells is negligible[END_REF]and Kuntner et al., 2010.

  to develop transporter-specific MRI contrast agents or SPECT tracers. Examples with re-purposed SPECT tracers such as [ 99m Tc]mebrofenin have illustrated the power of SPECT to study transporters in vivo, particularly in view of recent advances in SPECT methodology. Further research should be therefore directed toward the development of SPECT tracers as innovative transporter imaging probes with a broader clinical applicability. To establish the clinical benefit of transporter imaging, further studies in various disease settings with currently available or newly developed imaging probes are required. Nicolas Tournier received a grant from the French national research agency (ANR-16-CE17-0011-001). He thanks his colleagues from the Service Hospitalier Frédéric Joliot (part of the France Life Imaging network partly funded by the grant ANR-11-INBS-0006) for their work. Bruno Stieger is supported by grant #310030_166563 from the Swiss National Science Foundation (SNF) and by the SNF National Center of Competence in Research TransCure (University of Berne, Switzerland). Oliver Langer's work has been supported by the Austrian Science Fund (FWF) [grant numbers F 3513-B11, F 3513-B20, P24894-B24, I 1609-B24 and KLI-480-B30], the Lower Austria Corporation for Research and Education (NFB) [grant numbers LS12-006 and LS15-003] and the European Community's Seventh Framework Programme (FP7/2007-2013) [grant agreement number 201380]. Alexander Traxl and Martin Bauer are gratefully acknowledged for help with the preparation of the figures. The PET groups at the Department of Clinical Pharmacology and at the Division of Nuclear Medicine at the Medical University of Vienna and the imaging group at the Austrian Institute of Technology are acknowledged for their great cooperation over the past 15 years. Oliver Langer is a member of the Medical Imaging Cluster (MIC) at the Medical University of Vienna.

Figure legends Fig. 1 .

 legends1 Figure legends

Fig. 2 .

 2 Fig. 2. Chemical structures of selected imaging agents used to study drug transporters, including the SPECT tracer [ 99m Tc]mebrofenin (a) and the MRI contrast agent Gd-EOB-DTPA (gadoxetate) (b). Chemistry schemes for the synthesis of (R)-[ 11 C]verapamil by N-methylation of N-desmethyl verapamil using [ 11 C]methyl triflate (c) and 3 -[ 18 F]fluorocholic acid by nucleophilic 18 F-fluorination

Fig. 3 .

 3 Fig. 3. Kinetic model of the transport of [ 11 C]cholylsarcosine ( 11 C-CSar) from blood to bile. The

Fig. 4 .

 4 Fig. 4. PET summation images of rat (a) and human (b) brain obtained with the P-gp substrate radiotracer (R)-[ 11 C]verapamil at baseline and after administration of increasing doses of the third-

Fig. 5 .

 5 Fig. 5. Representative [ 11 C]glyburide PET data obtained in the liver of one non-human primate.

Fig. 6 .

 6 Fig. 6. Serial abdominal PET images recorded in mice (a) and humans (b) after intravenous injection of a microdose of [ 11 C]erlotinib (scan 1) and after injection of a microdose of [ 11 C]erlotinib

Figure 6

 6 Figure 6

Acknowledgement

Conflict of interest statement

The authors declare that there are no conflicts of interest.