Small Molecule PET Tracers for Transporter Imaging

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Small Molecule PET Tracers for Transporter Imaging Michael R. Kilbourn, PhD As the field of PET has expanded and an ever-increasing number and variety of compounds have been radiolabeled as potential in vivo tracers of biochemistry, transporters have become important primary targets or facilitators of radiotracer uptake and distribution. A transporter can be the primary target through the development of a specific high-affinity radioligand: examples are the multiple high-affinity radioligands for the neuronal membrane neurotransmitter or vesicular transporters, used to image nerve terminals in the brain. The goal of a radiotracer might be to study the function of a transporter through the use of a radiolabeled substrate, such as the application of 3-O-[11C]methyl]glucose to measure rates of glucose transport through the blood–brain barrier. In many cases, transporters are required for radiotracer distributions, but the targeted biochemistries might be unrelated: an example is the use of 2-deoxy-2-[18F]FDG for imaging glucose metabolism, where initial passage of the radiotracer through cell membranes requires the action of specific glucose transporters. Finally, there are transporters such as p-glycoprotein that function to extrude small molecules from tissues, and can effectively work against successful uptake of radiotracers. The diversity of structures and functions of transporters, their importance in human health and disease, and their role in therapeutic drug disposition suggest that in vivo imaging of transporter location and function will continue to be a point of emphasis in PET radiopharmaceutical development. In this review, the variety of transporters and their importance for in vivo PET radiotracer development and application are discussed. Transporters have thus joined the other major protein targets such as G-protein coupled receptors, ligand-gated ion channels, enzymes, and aggregated proteins as of high interest for understanding human health and disease. Semin Nucl Med ■■:■■–■■ © 2017 Elsevier Inc. All rights reserved.

Introduction

I

n a simple sense, a transporter is a protein that functions to move a substrate from one location to another. Most often, this is to move an ion or molecule across a lipid bilayer membrane barrier, where passive diffusion is minimal or limited by such an ionic or concentration gradient. The majority of transporters are located at epithelial barriers (eg, intestinal epithelia, hepatocytes, brain capillary endothelial cells, choroid plexus cells, kidney proximal tubule cells), but they are also found on intracellular membranes (vesicles, mitochondria, Golgi, vacuoles, endosomes).1,2 The movement of the substrate can be passive or active, involve cotransport or antitransport of other molecules or ions (which may involve a

Department of Radiology, University of Michigan Medical School, Ann Arbor, MI. Address reprint requests to Michael R. Kilbourn, PhD, Department of Radiology, University of Michigan Medical School, Ann Arbor, MI 48105. E-mail: [email protected]

http://dx.doi.org/10.1053/j.semnuclmed.2017.05.005 0001-2998/© 2017 Elsevier Inc. All rights reserved.

second transporter), and may or not require expenditure of energy (hydrolysis of adenosine triphosphate [ATP]). The molecular mechanism of solute transport remains unknown for the majority of transporters, but is in general might be described by an alternating access model, where the substrate binding site is exposed to one side, transits through an occluded state (not exposed to either side of membrane), and then exposed to the opposite side.3 The determination of the three-dimensional structures of these important proteins, defining the locations of substrate interactions, and delineating the mechanism of substrate movement are active areas of research as transporters are involved in many aspects of human health and disease. Mutations and variations in transporter genes are the basis of several inherited diseases, and an astonishing number of therapeutic drugs currently used in medicine are either substrates for or inhibitors of transporters.4,5 As examples, inhibitors of the neuronal membrane serotonin transporter (SERT/SLC6A4) such as fluoxetine (Prozac) and citalopram (Celexa) are commonly used in neurology and psychiatry, and inhibitors of the sodium-glucose cotransporter 1

ARTICLE IN PRESS M.R. Kilbourn

2 2 (SGLT2/SLC5A2) as a recent therapy for type 2 diabetes mellitus (eg, Farxiga). The numbers and types of molecules needing to be transported across membranes are very large, and there is a corresponding very large and diverse number of transporters expressed throughout the body. Hundreds of proteins that have been assigned as having transporter function are broadly divided into two large groups, the solute carrier (SLC) and ATP-binding cassette (ABC) families. As new proteins are being continuously identified and assigned to these two families, the numbers are continually changing. A comprehensive review of transporters has been recently published,6 with active links to the web-based details of individual transporters (IUPHAR/ BPS Guide to Pharmacology: http://www.guidetopharmacology .org). The SLC superfamily consists of 52 gene families and over 400 individual proteins. Assignment into a gene family is based on sequence similarity (sharing at least 20% identity) and likely transmembrane α-helical structures. However, members within a single family can have distinctly different functions, exemplified by the SLC22 family that includes transporters of both organic anions and cations. Furthermore, the functional selectivity or specificity of transporters can be shared by SLC families that are unrelated in the genetic sense: for example, no less than 10 different SLC families function in the accumulation of amino acids. Finally, transporters can act in concert, as exemplified by the system XC−, the cystine-glutamate antiporter that is a covalent combination of two separate transporters SLC3A2 and SLC7A11: one protein is responsible for transport function and the other for cell surface expression.7 The human ABC transporter proteins have been arranged into seven subfamilies, with 49 identified human ABC genes.8 All ABC transporters share similar structural characteristics, the most obvious being the binding site for ATP. As with the SLC transporters, individual members of the ABC transporter subfamilies may have different substrate selectivities (eg, ABCB1 for multidrug resistance and ABCB2 for peptide transport), and transport functions may be shared by members of different subfamilies (eg, ABCA3, ABCB1, and ABCC1 are all involved in multidrug resistance).

Transporter Nomenclature Transporters are a very large group of proteins, with very diverse structures and functions, and any review of transporters is furthermore complicated by the confusing nomenclature. The most precise nomenclature for transporters is that based on the gene assignment. Thus, for the SLC transporters, there are 52 families designated SLC1–SLC52, with individual family members given an additional identifiers (eg, SLC1A1, SLC1A2, etc). A similar nomenclature style is used for the ABC transporters: families are ABCA–ABDG, with individual gene members identified with an additional number (eg, ABCA1, ABCA2, etc). Although a simple nomenclature scheme, these names are entirely uninformative as to the function of any transporter, and furthermore as these identifiers are of only recent origin due to the advances in genetics, the majority of the older literature uses more descriptive if less precise transporter nomenclature.

A single example may suffice to show the variety of names that can be attached to a single transporter. The neuronal membrane reuptake of L-glutamate is critically important in mammalian brain function, and in the human brain is performed by multiple transporters; one of these is SLC1A2, also known in the literature as EAAT2 (excitatory amino acid transporter 2) and GLT1 (glutamate transporter 1). The latter two names are certainly more descriptive, although the differing numbers (1 vs 2) provide confusion! Just to add to that, the rodent equivalent of EAAT1 was historically given the nomenclature GLAST (glutamate/aspartate transporter). To provide the most accurate information, in this review, transporters will be identified when possible using both the descriptive name and abbreviation (eg, dopamine transporter or DAT) followed by the gene nomenclature (SLC6A3).

Transporters and In Vivo Imaging Transporters are significant targets for PET radiopharmaceutical development,9,10 but the role of transporters in PET radiotracer uptake and localization has perhaps not been fully appreciated: although not designed per se as transporter imaging agents, early PET radiotracers such as [ 18 F]fluoro-dihydroxyphenylalanine ([ 18 F]DOPA) 11 and 2-[18F]FDG12 required the obligatory participation of transporters (the large amino acid transporter LAT1/SLC7A5 and the SLC2 family of hexose transporters, respectively) for their uptake and distribution in the human body even though the applications of the radiotracers were to study dopamine biosynthesis and glucose metabolism. In fact, even when PET radiotracers were beginning to be developed that specifically targeted a neuronal membrane transporter in the brain (eg, dopamine transporter DAT/SLC6A3), they were still sometimes referred to as neuroreceptor imaging agents. It is remarkable that, of the 52 SLC families of transporters, 19 are involved as either direct targets for or facilitate the uptake of PET radiotracers (Table). The role of transporters in PET radiotracers is discussed here in relation to major organs of the bodies (eg, brain, heart, kidney) or tumors, with an emphasis on the SLC family of transporters, as these are the ones primarily responsible for the localization of radioactive tracers in the body. It should however be recognized that many of the transporters discussed are found throughout the body, in different tissue types (eg, [18F]FDG in brain, heart, and tumors), and the radiotracers that are dependent on transporters for binding or transport may have potential application in many different organs. It should also be recognized that all PET radiotracers (except [15O]water) are subject to metabolism and excretion, processes that involve transporters and that indirectly affect the distributions of radioactivity within the body.

SLC Transporters in the Brain The human brain has been the major target for in vivo PET radiopharmaceutical development since the early introduction of PET imaging equipment. Radiopharmaceutical development for brain imaging has always faced the challenge of the blood–brain barrier (BBB), a specialized membrane structure that functions to limit passage of polar,

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Table SLC Transporters Involved in PET Radiotracer Distributions as Either Mechanism of Initial Tissue Uptake or Directly as Biochemical Target SLC Gene SLC1 glutamate transporters SLC1A3 SLC1A2 SLC1A3 SLC1A5 SLC2 hexose transporters SLC2A1 SLC2A4 SLC2A5 SLC3 heteromeric amino acid transporters (with SLC7 proteins) SLC3A2 SLC5 sodium-dependent glucose transporters SLC5A1 SLC5A2 SLC5 sodium/iodide symporter SLC5A5 SLC5 choline transporter SLC5A7 SLC6 neurotransmitter transporters SLC6A2 SLC6A3 SLC6A4 SLC6A1 SLC6A11 SLC6A9 SLC6A14 SLC7 neutral amino acid transporters (with SLC3 proteins) SLC7A5 SLC7A11 SLC7A9 SLC10 sodium-bile acid co-transporters SLC10A1 SLC10A2 SLC11 proton-coupled metal ion transporters SLC11A1 SLC11A2 SLC15 peptide transporters SLC15A2 SLC16 monocarboxylate transporters SLC16A1 SLC16A2 SLC18 vesicular amine transporters SLC18A2 SLC18A3 SLC22 organic anion and cation transporters SLC22A1 SLC22A2 SLC22A6 SLC23 ascorbic acid transporters SLC23A1 SLC27 fatty acid transporters SLC27A1 SLC27A4 SLC29 equilibrative nucleoside transporters SLC29A1 SLC31 copper transporters SLC31A1 SLC38 sodium-dependent amino acid transporters SLC38A1 SLC44 choline transporters SLC44A1 SLC47 multidrug and toxin extrusion transporters SLC47A1 SLCO family of organic anion transporting polypeptides SLCO1A2 SLCO1B1 SLCO1B3 Examples of radiotracers are shown; see text for additional radiotracers.

Abbreviation

Substrates

Radiotracers

EAAT1 EAAT2 EAAT3 ASCT2

Glutamate Glutamate Glutamate Ala/Ser/Cys

None None None Anti-[18F]FACBC

GLUT1 GLUT4 GLUT5

Glucose Glucose

[18F]FDG, [11C]glucose, 3-O-[11C]Me-glucose [18F]FDG, [11C]glucose [18F]fluorofructose

XC-

Glutamate

[18F]fluoropropyl-glutamate

SGLT1 SGLT2

Glucose Glucose

α-methyl-4-[18F]glucose

NIS

Iodide

[18F]BF4,[18F]SO3-

CHT1

Choline

[11C]choline, [18F]fluorocholine

NET DAT SERT GAT1 GAT3 GlyT1 ATB0, +

norEPI, DA norEPI, DA Serotonin GABA GABA Glycine Beta-alanine

[11C,18F]reboxetines [11C,18F]phenyltropanes [11C,18F]diarylsulfides [18F]diarylalkyl-nipecotic acids [18F]triarylalky-nipecotic acid [18F]MK-6577, [11C]RO5013853

LAT1 XCb0,+AT

Phenylalanine Glutamate Amino acids

[11C,18F]amino acids, [18F]FDOPA

NTCP ASBT

Taurocholic acid Glycodeoxycholate

[11C]methyl-Taurocholic acid [11C]methyl-Glycocholic acid

NRAMP1 DMT1

Fe2+,Mn2+ Cu2+,Fe2+,Mn2+

PEPT2

Dipeptides

[11C]GlySar

MCT1 MCT2

Pyruvate, lactate Pyruvate

[11C]pyruvate

VMAT2 VAChT

Monoamines Acetylcholine

[11C,18F]tetrabenazines [18F]FEOBV,[18F]VAT

OCT1 OCT2 OAT1

Organic cations Organic cations Organic anions

[11C]choline [11C]choline [18F]FAMT

SVCT1

Ascorbic acid

[11C]ascorbic acid

FATP1 FATP4

Palmitate Palmitate

[11C,18F]fatty acids [11C,18F]fatty acids

ENT1

Nucleosides

[11C]thymidine, [18F]FLT

CTR1

Copper

64

SNAT1

Ala, Met

[11C]methionine

CTL1

Choline

[11C]choline

MATE1

Creatine

[11C]metformin

OATP1A2 OATP1B1 OATP1B3

Anionic drugs Anionic drugs Anionic drugs

[18F]pitavastatin

52

Mn2+ Mn2+, 64Cu2+

52

Cu2+

ARTICLE IN PRESS M.R. Kilbourn

4 hydrophilic molecules from the blood to the brain. Obviously, the BBB limits but does not exclude all molecules from entry into the brain tissues. The major mechanism for passage through the BBB and into the brain tissues are a myriad of SLC transporters with disparate substrate specificities,13 such that the brain can be supplied with the requisite molecules for energy metabolism, synthesis (proteins, phospholipids, neurotransmitters), and maintaining ion balance, among other functions. PET radiotracer development for the brain has involved two broad classes: (1) those that involve a transporter at the luminal (blood side) of the BBB for initial uptake, although the eventual physiological process to be studied occurs in cellular elements in the brain tissues, and (2) those that are lipophilic and enter the brain by passive diffusion where they interact with one or more transporters on or within cells. For studies of specific transporters in the BBB, no radiotracers have been developed that are bound but not transported. For radiotracers in (1), their uptake and localization can be impacted by changes in transporter number or function, and this must always be remembered. For radiotracers in (2), the initial brain uptake may be simple diffusion, but high lipophilicity introduces the challenge of nonspecific binding. Finally, just to further complicate radiotracer design, the BBB is also equipped with SLC transporters that operate bidirectionally and can pump molecules out of the brain and back into the plasma, along with promiscuous transporters of the ABC family (termed multidrug transporters) that function as extrusion transporters (discussed later). Despite these challenges to radiotracer design, an extensive list of compounds have been radiolabeled and used in studies of the central nervous system, with a significant portion of these either specifically targeting transporters or requiring the actions of transporters for their uptake and distribution in the brain tissue.

Brain Metabolic Radiotracers The primary energy source for the brain is glucose, and carbon11 labeled glucose and the analog 2-[18F]FDG have both been used for imaging of cerebral glucose metabolism. Syntheses of [11C]glucose are challenging, and the modeling of its in vivo pharmacokinetics difficult, and thus it has been essentially completely replaced by [18F]FDG. [18F]FDG is widely used across the globe for neurologic PET studies, and the application of [18F]FDG is generalized to quantitatively image changes in glucose metabolism. What is underappreciated is that the brain uptake of radiolabeled hexose sugars, including [18F]FDG, is dependent on the function of transporters belonging to the sodium-independent glucose transporters of the SLC2 family, with the most important being GLUT1/ SLC2A1 in the endothelial cells of the BBB and GLUT3/ SLC2A3 on neuronal membranes.14 Of course, [18F]FDG is not exactly glucose, and there are subtle differences in the rate of transport between glucose and [ 18 F]FDG. 15 For direct studies of the GLUT-mediated transport of glucose, radiotracers such as 3-O-[ 11 C]methyl-D-glucose 16 or 3-[18F]fluoro-deoxyglucose17 are necessary, which are transported through membranes but cannot be phosphorylated by intracellular hexokinase. The widespread use of [18F]FDG as a measure of glucose metabolism is dependent on the as-

sumption that, in normal brain tissue, the rate of phosphorylation of glucose by cellular elements is significantly greater than the transport of glucose through the BBB, and thus transport is not considered the rate-limiting step in the accumulation of radioactivity. Movement of glucose in the brain is however more complex, as numerous other sodium-independent glucose transporters (SLC2A4-8) and sodium-dependent glucose transporters (SGLT1 (SLC5A1) and SGLT2 (SLC5A2)) are expressed in the human brain.14 The role of the SGLTs in normal and pathologic conditions is yet to be fully studied, and for this purpose, the SGLT2 substrate alpha-methyl-4-[18F]FDG has been recently prepared and shown to accumulate in the rat brain.18 Finally, and fortunately for the field of PET imaging, [18F]FDG is not a substrate for the extrusion by ABC transporters p-glycoprotein (pgp/ACCB1) and breast cancer resistance protein (BCRP/ABCG2)19: as will be discussed later, the ABC transporters can complicate brain PET radiotracer design. Glucose is not the only potential energy substrate used by the brain. As an alternative, simpler molecules such as pyruvic acid, β-hydroxybutyrate, and acetoacetic acid (commonly termed ketone bodies) can be metabolized under certain physiological conditions. Each of these small molecules has been radiolabeled with carbon-11 and used in human PET studies.20-22 The brain uptake of these keto acids is dependent on monocarboxylate transporters (MCT1/SLC16A1) at the endothelial cell level of the BBB. As the enzymes involved in aerobic pyruvate metabolism (eg, pyruvate dehydrogenase) are located inside the mitochondria, labeled pyruvate must furthermore be transported across the inner mitochondrial membrane; this is accomplished by the heterooligomeric complex of proteins termed the mitochondrial pyruvate carriers (MPC1 and MPC2), although these have not been clearly characterized as SLC proteins.23 Thus, radiolabeled pyruvate, hydroxybutyrate, and acetoacetic acids are used as metabolic tracers, but the brain uptake and retention are dependent on the integrity of one or more transporter systems. Finally, several simple carboxylic acids ([11C]butyrate, 11 [ C]hexanoate, [ 11 C]octanoate) and longer chain acids ([11C]palmitate, [11C]docosahexanoic acid [DHA]) have been synthesized and studied for brain uptake as potential markers of metabolism. All of these are transported at the BBB by proteins such as the fatty acid transport protein FATP1/SLC27A1.

Brain Transport of Amino Acids Amino acids are examples of molecules efficiently excluded by the BBB: but as amino acids are the building blocks of proteins, serve as precursors for the synthesis of important neurotransmitters (eg, monoamines), and can even be energy sources, it is critically important that they are readily available to the brain tissues. Given the variety of amino acid structures (neutral, acidic, and basic), it is perhaps not surprising there are multiple transporters in the BBB that move amino acids into the brain extracellular space,13 and several are key to PET radiotracer distributions into the brain. The aromatic amino acids tryptophan and tyrosine are the precursors for the biosynthesis of the monoaminergic neu-

ARTICLE IN PRESS PET Tracers for Transporters rotransmitters serotonin, dopamine, and norepinephrine. Conceptually, radiolabeled forms of these amino acids should be readily taken up by transporters in the BBB (primarily the LAT1/SLC7A5), and once inside the brain used for neurotransmitter syntheses: storage of the newly formed monoamines in vesicles would provide a mechanism for tissue localization and retention. In practice, such studies are generally done using radiolabeled forms of tyrosine (eg, 3-[ 18 F]fluorotyrosine) 24 or dihydroxyphenylalanine (eg, 6-[18F]fluoroDOPA or [11C]DOPA)11,25 for dopamine synthesis, and derivatives of tryptophan (eg, [ 11 C]α-methyltryptophan)26 for serotonin. So although the intent of these radiotracers is to provide measurements of the rate of syntheses of the ultimate neurotransmitters (eg, [18F]fluoroDOPA to form [18F]fluorodopamine as an estimate of dopamine synthesis), the proper functioning of the LAT1 transporter is crucial to the distribution of the radiotracer into the brain. The transport of [18F]fluoroDOPA should thus be subject to competition by other large amino acids in plasma, such as phenylalanine, and there is evidence for such in a disease state (phenylketonuria).27 Furthermore, the tissue retention of radioactivity after [ 18 F]fluoroDOPA administration is attributed to vesicular trapping of the newly synthesized [18F]fluorodopamine: that process requires the participation of yet another transporter, the vesicular monoamine transporter type 2, which will be discussed in a later section. Many additional amino acids and derivatives have been radiolabeled,28 and brain uptake of each has depended on the initial transporter-mediated passage through the epithelial cells of the BBB. Most radiolabeled amino acids were intended to measure a biochemical process other than transport (such as neurotransmitter synthesis, or protein synthesis); to isolate and measure transporter function specifically, it was necessary to design an amino acid that would be transported but not further metabolized in the brain, such as [11C]aminocyclohexanecarboxylic acid ([11C]ACHC). This nonmetabolized radiotracer could then be used to demonstrate competitive inhibition of LAT1-dependent amino acid transport through the BBB.29 Thus, although the goal of using a radiolabeled amino acid may be to study protein synthesis, or neurotransmitter synthesis, or formation of important molecules such as glutathione, the initial uptake of virtually every radiolabeled amino acid involves participation of one or more SLC transporter at the BBB.

Brain Neurotransmitter Transporters A significant portion of the effort in PET radiopharmaceutical development has been directed toward the brain transporters for neurotransmitters. These transporters are predominantly located at the neuronal membrane (neuronal membrane transporters) or on intracellular storage vesicles (vesicular neurotransmitter transporters) of neurons and are thus inside the BBB: in this instance, radiotracers have been designed to be sufficiently lipophilic molecules capable of passive diffusion through the BBB and do not involve additional transporters for initial uptake from the blood. In contrast to the radiotracers for metabolism, or the amino acids used

5 for syntheses of neurotransmitters or proteins, radiotracers designed for the brain neurotransmitter transporters have been uniformly high-affinity ligands that are bound to the proteins but are not transported. For the monoamine neurotransmitters dopamine, norepinephrine, and serotonin, there are three selective neuronal membrane transporters: the dopamine transporter (DAT/ SLC6A3), norepinephrine transporter (NET/SLC6A2), and the serotonin transporter (SERT/SLC6A4). These are all members of the same family and share similar molecular structures30: the challenges for radiotracer design have been the need for adequate lipophilicity (be able to pass through BBB but not exhibit high nonspecific binding) and that the three monoamine transporters show poor specificity for many of the substrates and inhibitors that are potential molecular structures for radiolabeling. Nevertheless, an extraordinarily large number of radiotracers have been synthesized and evaluated in animals as potential DAT and SERT radioligands, and a smaller but still significant number of these advanced into human studies. Radiotracer development for the NET has been more problematic and has resulted in a smaller number of candidate radioligands, of which only few have seen evaluation in human studies. Radioligand development for the DAT has predominantly focused on molecules based on the tropane ((1R,5S)-8-methyl8-azabicyclo[3.2.1]octane) scaffold, initially derived from the structure of cocaine, which itself had been radiolabeled with carbon-11 and used in early human studies. Most useful have been the 3-phenyltropanes exemplified by [11C]CFT (WIN 35,428) and numerous derivatives with [18F]fluoroalkyl substituents (Fig. 1). The tropane structure has two convenient locations for radiolabeling, using either N-alkylation or O-alkylation reactions, and thus numerous potential radiotracers have been synthesized: a recent review lists 46 different tropanes synthesized as potential DAT radioligands.31 The list also includes 3-phenyltropanes with aryl [18F]fluoride substituents, and the continued development of novel methods to achieve high-specific [18F]fluorination of unactivated phenyl rings32 may make those radiotracers of increased interest. Most if not all of the many tropane-based DAT radioligands provide successful in vivo imaging in animal or human brain, with the differences being in specificity (DAT over SERT and NET), pharmacokinetics, and potential metabolism to interfering BBBpenetrating metabolites. Tropanes have not been the only scaffold used for DAT radioligand development. Diverse structures such as the diarylalky piperazines (1-[2-[bis(4-fluorophenyl)methoxy ]ethyl]-4-(3-phenylpropyl)piperazine, GBR 12909),33 the PCP analog BTCP (1-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine),34 nomifensine,35 and methylphenidate36 have been radiolabeled with carbon-11 or fluorine-18; of these, only [11C]methylphenidate has seen extensive application in human PET studies of neurologic diseases. For the serotonin neuronal membrane transporter (SERT/ SLC6A4), there has been a similarly large effort at radiotracer development. Early efforts centered on radiolabeling of clinically used SERT inhibitors known as selective serotonin uptake inhibitors, such as fluoxetine, paroxetine, citalopram, and

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M.R. Kilbourn

Figure 1 Structures of representative tropane, diphenylsulphide, and morpholine based radioligands used for PET imaging of the neuronal membrane transporters for dopamine (DAT), serotonin (SERT), and norepinephrine (NET).

sertraline. None of these proved optimal for in vivo imaging, largely due to the high lipophilicity and excessive nonspecific binding. Greater success was achieved using two very different scaffolds that are not part of clinical pharmacology: 3-phenyltropanes and diarylsulfides. A significant number (at least 19) of derivatives of the 3-phenyltropane structure have been prepared in the search for a specific SERT radioligand: this has proved very challenging, as no radiotracer to date has been demonstrated to be specific for the SERT, and most have some (and in many cases significant) affinity for the DAT or the NET.37 Better success has been achieved with the diaryl sulfide structure: the early radiotracer, and still in widespread use today, was [11C]DASB with a high affinity (Ki = 1 nM) for the SERT and excellent selectivity (Ki values for DAT and NET are both >1 micromolar) (Fig. 1). The success of [11C]DASB prompted the synthesis of numerous derivatives labeled with carbon-11 or fluorine-18, of which at least 31 different molecules have been synthesized37: some have even higher SERT affinity or specificity, but [ 11 C]DASB remains the predominantly used radiotracer for human PET studies. The third member of the monoamine transporters, the NET/ SLC6A2, has proven the most challenging. Early attempts using carbon-11 labeling of desipramine and nisoxetine38 were unsuccessful due to the high lipophilicity of the molecules. Many of the 3-phenyltropane derivatives synthesized in the search for DAT and SERT radioligands show moderate to high NET affinity, but none exhibited the needed specificity for the NET over the DAT and SERT. However, recently a new derivative [ 11 C]NS8880 with good affinity and selectivity was reported; in vivo studies are encouraging.39 Most recent NET radiotracer development has centered around the structure of reboxetine (Fig. 1), a morpholine-substituted analog of nisoxetine.40 Numerous carbon-11 and fluorine-18 radioligands derived from reboxetine have been synthesized and examined in animals or humans, but the successful application of NET radioligands in human PET studies has proven complicated. The low and widespread distribution of the NET in the human brain challenges methods for pharmacokinetic modeling of the PET imaging data, as identification of a region with little or no specific binding (to estimate nonspecific distribution) has been difficult. Second, many of the reboxetine radioligands are metabolized to form interfering brain-

penetrant metabolites; in an attempt to reduce the formation of metabolites, substitution with deuterium has been applied to provide radiotracers with slower rates of peripheral metabolism.41 Other members of the SLC6 transporter family found in the human brain include the neuronal membrane transporters for GABA (GAT-1/SLC6A1 and GAT-3/SLC6A3) and glycine (GlyT-1/SLC6A9 and GlyT-2/SLC6A5),42 but PET radiotracer development for these has proven much more challenging than for the monoamine transporters. For the GAT-1, there are few options: the only inhibitor used as a human drug is tiagabine,43 a nipecotic acid derivatized with a diphenylalkyl group to provide sufficient lipophilicity to allow passage through the BBB. A few potential fluorine-18 labeled nipecotic acid derivatives have been prepared (Fig. 2) and were evaluated in animals, but all have shown low brain uptake and as yet no successful human PET imaging agent has been achieved.44,45 A similar effort to develop a fluorine-18 labeled GAT-3 radioligand was unsuccessful.46 For the glycine transporter (GlyT1/SLC6A9), the efforts have been more fruitful; a small series of carbon-11 and fluorine-18 labeled GlyT1 inhibitors have been prepared and evaluated in nonhuman primates or humans.47-53 Interestingly, the compounds chosen for radiolabeling are fairly large multiring structures (as an example, [18F]MK-6577; Fig. 2), and they look nothing like the simple amino acid neurotransmitter, so one might proposed these radioligands are binding to a site on the transporter that may not be the same as that which binds the very small molecule glycine. Nevertheless, they provide in vivo images that have been described as consistent with the distribution of the GlyT1 transporter in human brain in vitro. There are other neuronal membrane transporters for which no successful in vivo radiotracers have been developed. For glutamate (excitatory amino acid transporters EAACT1/ SLC1A3, EAACT2/SLC1A2, EAACT3/SLC1A1), a paucity of lipophilic high-affinity inhibitors that could serve as candidates for radiolabeling have stymied development of radiotracers.54 Research into EAACT inhibitors has yielded high-affinity ligands, but they retain carboxylic acid functional groups and thus are poor candidates for in vivo radioligand development.55 Acetylcholine is also an interesting challenge, as the classic representation of the cholinergic system shows that acetylcholine is efficiently hydrolyzed by

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Figure 2 Structures of radioligands prepared for PET imaging of the neuronal membrane GABA (GAT1) and glycine (GlyT1) transporters in the brain.

acetylcholinesterase in the synapse and the product of the enzyme reaction (choline) is taken back up into the neuron for de novo synthesis of acetylcholine: no mention is typically made of a neuronal membrane acetylcholine transporter. A recent study challenges that model, providing evidence for a unique acetylcholine transport mechanism in the rat brain56: if and when the transporter involved is identified, perhaps specific ligands will be identified and attempts can be made to develop the corresponding in vivo radioligands. In the meantime efforts in the cholinergic system have centered on potential radioligands for the high-affinity choline transporter CHT1/ SLC5A7, but such have been hampered by, again, a lack of chemical scaffolds. Early attempts to radiolabel derivatives and analogs of hemicholinium-3, the classic CHT1 inhibitor, proved unsuccessful due to the polarity of the molecules.57,58 More recently, a new scaffold for high-affinity CHT1 inhibitors was reported,59 with such structures as ML352 (Fig. 3) proposed to bind to a site distinct from the acetylcholine binding site; as seen for the glycine transporter inhibitors of interest

Figure 3 Structures of candidate radioligands for PET imaging of the high-affinity choline transporter CHT1.

as radioligands, such potential CHT1 ligands bear little resemblance to the endogenous transporter substrate acetylcholine.60 Carbon-11 labeling of ML352 was accomplished, but to date good brain uptake has not been achieved.61 The second major group of brain neurotransmitter transporters is the vesicular transporters.62 Located on the storage vesicles of the neuron, these proteins are responsible for movement of neurotransmitters into the vesicle against a concentration gradient, to achieve the high concentrations of neurotransmitter molecules needed in vesicles before stimulated release. A single vesicular transporter, the vesicular monoamine transporter type 2 (VMAT2/SLC18A2), is found in all monoaminergic nerve terminals and transports dopamine, serotonin, norepinephrine, and a variety of other basic lipophilic amines. For other neurotransmitters (GABA, glutamate, acetylcholine), there are distinct vesicular transporters. For radiotracer development aimed at the VMAT2, compound choices were actually very few: reserpine, a very highaffinity but essentially irreversible inhibitor; ketanserin, a moderate affinity VMAT2 inhibitor with affinity also for serotonin receptors; and tetrabenazine (TBZ), a benzoisoquinoline developed in the 1950s and currently in clinical use for treatment of hyperkinetic movement disorders. Fortuitously, TBZ was found to have excellent physiochemical properties (log P = 2.1) and pharmacology (no affinity for other vesicular or neuronal membrane transporters) and could be readily labeled with carbon-11.63 Successful in vivo imaging64 of the VMAT2 in human brain using [11C]TBZ was followed by several years of effort to identify and radiolabel the highest affinity stereoisomer of the pharmacologically active metabolites, culminating in the optimal derivative 2R,3R,11bRdihydrotetrabenazine (DTBZ, Fig. 4). Success of [11C]DTBZ imaging led to development of fluorine-18 analogs,65 including [18F]AV-133 (9-O-[18F]fluoropropyl-DTBZ), which has reached human studies.66 The VMAT2 radioligands are an excellent example of lipophilic molecules that need to passively permeate not just one, but two membrane barriers, as the binding site for [11C]DTBZ is likely on the inside surface of the vesicle membrane. In contrast to the VMAT2 radiotracer development, which was based on a clinically used drug with decades of human use experience, the design of radiotracers for the vesicular

ARTICLE IN PRESS M.R. Kilbourn

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Figure 4 Structures of radioligands used for human PET imaging of the vesicular monoamine (VMAT2) and acetylcholine (VAChT) transporters.

acetylcholine transporter (VAChT/SLC18A3) has been much more difficult and protracted. There is no VAChT inhibitor in clinical drug use: inhibition of the VAChT has been found to have serious detrimental pharmacologic effects in animals. All of the radiotracer development has been based on the structure of vesamicol (2-(4-phenylpiperidino) cyclohexanol (AH5183), a compound identified in the late 1980s as a highaffinity inhibitor of vesicular acetylcholine transport.67 Early attempts to use vesamicol as the radioligand were unsuccessful, leading to years of research into derivatives (benzovesamicols, decalinvesamicols) and analogs (eg, trozamicols, piperazines, spirovesamicols) that would exhibit the desired combination of selectivity for the VAChT (many derivatives of vesamicol have measureable affinity for sigma receptors), acceptable toxicity, metabolic stability, and pharmacokinetics.68-71 Finally, after decades of development, human studies began with two fluorine-18 labeled VAChT benzovesamicol radioligands, [18F]FEOBV72 and [18F]VAT73 (Fig. 4). For the remaining major neurotransmitters, glutamate and GABA, there are specific vesicular transporters but no PET radioligands. Although the vesicular GABA transporter (VGAT/ SLC32A1: also termed the vesicular inhibitory amino acid transporter, VIAAT) has been identified,74 there are no highaffinity ligands known upon which to build a radiotracer strategy. For the vesicular GLTs (VGLUT1/SLC17A6, VGLUT2/ SLC17A7, VGLUT3/SLC17A8), the only known high-affinity inhibitors are complex dye structures75 such as Brilliant Yellow (Fig. 5).76 Such structures present a substantial challenge to radiopharmaceutical chemists, but perhaps one day an analog will be designed, in much the same way that the histochemical dye thioflavin-T was transformed into the PET radioligand for beta-amyloid, [11C]PIB. The various PET radioligands for the DAT, SERT, NET, and VMAT2 have now seen widespread applications in human studies: radioligands for the VAChT are being studied at multiple institutions. In general, interpretation of in vivo binding

has been that radiotracer distributions represent concentrations of presynaptic terminals associated with specific neuronal populations (eg, DAT for dopamine, VMAT2 for monoamines in general). That, of course, is a simplification of neurochemistry. A single transporter can be found on different neuron types, and also be present on glial cells, as is described for the glycine transporter GLYT1. The numbers of transporters can be regulated at both the transcriptional and translational steps, and the location and functional status regulated by post-translational processing (eg, phosphorylation) and intracellular trafficking. 77 Multiple neuronal membrane transporters78 or combinations of vesicular transporters (VGLUT has been found to co-localize with VMAT2, VAChT, and VGAT)79 can be found in a single neuron. These issues do not detract from the remarkable value of in vivo radiotracer imaging for studying changes in neurochemistry in disease states, but do point out that biology is infinitely more complex than one can sometimes imagine.

Other Brain SLC Transporters Other transporters located in brain tissues have been targeted for PET radiotracer development, and it can be expected

Figure 5 Structure of Brilliant Yellow dye, a high-affinity inhibitor of the vesicular glutamate transporter (VGLUT1). The highly polar structure presents challenges to PET radioligand design for brain imaging of the VGLUT1.

ARTICLE IN PRESS PET Tracers for Transporters that additional transporters will be of interest in tracer development for the future. Benzyl [11C]hippurate, a simple ester, was prepared for studies of the organic anion transporter 3 (OAT3/SLC22A8); the radiotracer is lipophilic and enters the brain passively through the BBB, and once inside is hydrolyzed to [11C]hippuric acid, which cleared from the brain in proportion to OAT3 activity.80 Evidence for this mechanism was shown using OAT3 knockout mouse studies. For studies of the drug/H+-antiporter, [11C]diphenhydramine has been synthesized and investigated in vivo in rodents. 81 The monocarboxylate transporter 1 (MCT1/SLC16A1) is expressed on the luminal side of BBB endothelial cells, and primarily functions to transport lactate and pyruvate into the brain. In an attempt to image potential binding sites for gamma-hydroxybutyrate, a proposed neurotransmitter and clinically used controlled substance, a carbon-11 cyclic analog labeled 3-hydroxycyclopent-1-enecarboxylic acid was prepared: lack of brain uptake was attributed to an unexpected failure of the radiotracer to be transported by the MCT1 at the BBB, despite prior evidence for saturable gammahydroxybutyrate transport.82

Heart Transporter Imaging A second major emphasis of PET radiopharmaceutical development has been for imaging biochemistry of the heart, and there are examples of radiotracers that either use transporters as part of their tissue uptake, or target the presence or function of a transporter directly.

Heart Metabolic Radiotracers Studies of heart bioenergetics using radiolabeled forms of glucose ([11C]glucose) or glucose derivatives ([18F]FDG), radiolabeled fatty acids (eg, [11C]palmitate or fluorine-18 labeled fatty acids83,84), or short-chain acids (eg, [11C]acetate) all involve a role for specific transporters. As in the brain, the heart uptake of glucose and glucose derivatives is dependent on the glucose transporters (principally GLUT4/ SLC2A4). The uptake of long-chain fatty acids (eg, [11C]palmitate) is facilitated by any of several fatty acid transport proteins, including the fatty acid transport proteins (FATP1-6/SLC27A1-6), the fatty acid translocase protein CD36, and fatty acid binding proteins. There is a complex relationship between glucose and fatty acid transport depending on the energetic demands of the tissues and substrate availability85; both the glucose and the proteins involved in fatty acid transporter are regulated proteins, with functional expression controlled by trafficking between the cell membrane and the cellular cytosol.86 The short-chain acid acetate is transported by the monocarboxylic acid transporter (MCT1/ SLC16A1), but the metabolism of [11C]acetate is less complex than for glucose or fatty acids, and the rate of clearance from heart tissues reflects a measure of oxidative metabolism.87 Thus, although the heart does not have the same tissue:plasma membrane barrier as the brain, uptake of radiolabeled metabolic substrates into cardiomyocytes requires and is dependent on action of transporters at the cardiomyocyte membrane,

9 irrespective of the eventual metabolic fate of the radiotracer inside the cell.

Heart Neurochemical Radiotracers The functioning of the heart tissues is dependent on the actions of two neurochemical systems, termed the sympathetic (adrenergic) and parasympathetic (cholinergic) systems. Of these the sympathetic nervous system of the heart has been extensively studied using PET radiotracers that directly involve one or both of the neuronal membrane norepinephrine transporter (NET, SLC6A2) and vesicular monoamine transporter type 2 (VMAT2, SLC18A2).88,89 In contrast to the brain, where high-affinity, nontransported radioligands for the NET or VMAT2 have been used, radiotracer development for the heart has been based on compounds that are functionally transported. The clinically used, single-photon emitting radiopharmaceutical m-iodobenzylguanidine (mIBG), originally developed for imaging of the adrenal glands and neuroendocrine tumors, exhibits high heart accumulation due to uptake from the blood by the NET followed by VMAT2-mediated storage in intracellular vesicles. Numerous carbon-11 and fluorine-18 labeled radiotracers based on the benzylguanidine structure have been subsequently synthesized and studied in animals or humans: all successfully image the heart, but with differences in pharmacokinetics and metabolism.90-93 As alternatives to the guanidine-based imaging agents, a number of PET radiotracers structurally related to the endogenous neurotransmitter norepinephrine have been synthesized. The most widely used has been [ 11 C]mhydroxyephedrine (HED), which along with the fluorine18 analogs 6- or 4-[18F]fluorometaraminol94,95 function as false neurotransmitters; these radiotracers are resistant to metabolism, are efficiently taken up by the NET, and once inside the heart neurons are efficiently transported into the storage vesicles by the VMAT2. The efficiency of these transport processes is such that uptake and retention of [11C]HED are essentially irreversible: the tracer however provides a useful index of adrenergic innervation in human heart. Radiotracers identical to or more closely related to the endogenous norepinephrine include [ 11 C]phenylephrine, 6-[ 18 F]fluoronorepinephrine, 6-[ 18 F]fluorodopamine, [11C]octopamine and [11C]epinephrine.96,97 The heart uptake and retention of these radiotracers involve the same two transporters (NET and VMAT2), but also to varying extent the actions of the enzymes catechol O-methyl transferase (COMT) and monoamine oxidase (MAO) (Fig. 6). Interpretation of heart uptake and kinetics of these radiotracers is thus more complex, but they offer insights into the roles of all of the components of the system (transporters and enzymes) involved in norepinephrine uptake, storage, and metabolism. The development of radiotracers targeting the parasympathetic (cholinergic) system of the heart has proven less successful. Studies using the VAChT radioligand [18F]FEOBV have demonstrated specific binding in heart tissues,98 but the very low levels of specific radiotracer localization have not supported movement of the radiotracer into human cardiac studies.

ARTICLE IN PRESS M.R. Kilbourn

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Figure 6 Structures of substrates radiolabeled for study of the adrenergic innervation of the human heart. Note radiotracers are all transported, but differentially affected by metabolism by monoamine oxidase (MAO) and catechol-Omethyltransferase (COMT).

Transporters in Other Organs Transporters have been targeted for radiotracer development in several other organs and tissues of the body, with most efforts aimed at the kidney, liver, and intestines. The hepatocytes in the liver express a number of transporters involved in the clearance of drugs and metabolites from the blood into the bile. The most studied using PET radiotracers are the organic cation transporter OCT1/SLC22A1 and the family of organic anion transporter proteins (OATPs/SLCO),99 and compounds of diverse structures have been radiolabeled and evaluated as in vivo imaging agents. A number of these are radiolabeled forms of clinically used drugs (eg, [11C]metformin, [11C]glyburide), and it can be expected that use of the radiotracer in humans should move forward: [11C]TIC-Me is an example however of a novel compound prepared as a radioligand for another target (prostacyclin receptor PGI2) that has progressed to human PET studies of its transport in liver. Using “click” chemistry, a recent development in fluorine18 chemistry that allows rapid radiolabeling of molecules by formation of fluorine-18 labeled triazole derivatives, radiotracers based on the bile acids cholic acid, deoxycholic acid, and lithocholic acid (LCATD) were prepared: one, [18F]LCATD, was used to image OATP1B1-dependent hepatobiliary excretion in an animal model.100 In a related approach, carbon11 labeled N-methyl-glycocholic acid and N-methyl-taurineconjugated bile acids were synthesized and studied as radiotracers for the sodium/taurocholate cotransporting polypeptides (ASBT/SLC10A2 and NTCP/SLC10A1).101,102 The biggest challenge to hepatic transporter imaging is that, to date, current PET radiotracers lack specificity: most are not only substrates for multiple organic anion or cation transporters, but in many cases also for other hepatic transporters including the multidrug and extrusion transporter (MATE1/ SLC47A1), and the ABC extrusion transporters MRP2/ ABCC2, pgp/ABCB1, and BCRP/ABCG2. The design of novel, specific radiotracers for many of these transporters remains a significant and unmet challenge. Numerous transporters (organic anion and cation transporters, multidrug and extrusion transporters, peptide

transporters) are involved in the movement of substrates across the membrane of the proximal tubules of the kidneys, and function to either move molecules out of the blood and into the urine, or for recovery of molecules from the urine. Many of the same transporters found in the liver are also found in the kidney, and thus not surprisingly radiotracers such as [11C]metformin (for OATP transporters) are also of interest in the kidney. The dipeptide radiotracer [ 11 C]glysar (glycylsarcosine) was developed as a substrate for the protoncoupled oligopeptide transporter type 2 (PEPT2/SLC15A2), and the specific uptake into the cortex of the kidney confirmed using PET imaging of PEPT2-knockout mice.103 Finally, imaging of transporters in the pancreas has seen much recent attention, given the growing incidence and economic impact of diabetes. However, investigations into pancreatic transporters have all used radiotracers that were initially developed for imaging in other organs.104 Radiolabeled amino acids ([11C]hydroxytryptophan and 5-(2-[18F]fluoroethoxy)L-tryptophan) localize into the pancreas, dependent on the action of the LAT1 transporter, but the localization did not correlate with beta-cell density.105 Radioligands originally developed for imaging of vesicular monoamine transporter 2 (VMAT2/SLC18A2) in the brain, such as [11C]DTBZ and [18F]AV-133, are being evaluated in humans as pancreatic beta-cell imaging agents, with controversial results.106 Finally, 4-[18F]fluorobenzyltrozamicol, a radioligand for the vesicular acetylcholine transporter VAChT/ SLC18A3 (developed for brain studies) has been examined for potential imaging applications in the pancreas.107 All of these are, in a way, re-purposing of existing PET radiotracers for pancreatic imaging purposes: the design of pancreasspecific, transporter-specific radiotracers has not been attempted yet.

Transporters in Tumors The application of PET and radiotracers in oncology is the most widespread clinical use of this technology, driven by the potential for improving the detection and characterization of tumors throughout the body. Tumor cells can be different from

ARTICLE IN PRESS PET Tracers for Transporters

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Figure 7 Structures of radiolabeled pyrimidines used for imaging of tumor proliferation.

normal tissues in a wide variety of biochemical processes, but the ones that have been most important for PET radiotracer development have been those associated with the enhanced growth and differentiation of tumor cells: increased metabolism, cell protein or phospholipid synthesis, and DNA synthesis.108,109 An increased metabolic demand is a well-characterized hallmark of tumor cells, most evident in an accentuated metabolism of glucose. High tumor uptake of glucose is the mechanism behind the success of [18F]FDG, the analog which is efficiently transported (GLUT1/GLUT4, SLC2A1/4) into and then trapped (after phosphorylation) by tumor cells. Tumor imaging with [18F]FDG is an accepted part of clinical medicine worldwide: the increased uptake of FDG into most tumors has made it useful not only for detection of primary tumors and metastases, but also for evaluation of tumor treatment response and post-treatment re-occurrence. Although [18F]FDG is described as a marker of glucose metabolism, it should be kept in mind that its localization required the glucose transporters. Although [18F]FDG may be the dominant radiotracer for tumor imaging, alternative metabolic substrates have been radiolabeled and employed in oncology. Simple carboxylic acids such as [11C]acetate, [11C]pyruvic acid, and [11C]acetoacetic acid enter cells through the monocarboxylate transporter (MCT1/SLC16A1) and thence into the mitochondria (via the mitochondrial transporters) where they can enter the Krebs cycle for energy production. All have been investigated as tumor imaging radiotracers in humans.110 Under certain metabolic conditions, tumors (and normal cells) can use glutamine as an energy source, through the conversion first to glutamate, then to α-keto-glutarate and entry into the Krebs cycle for ATP generation. Radiolabeled glutamine is thus an amino acid studied not just for protein synthesis, but also for metabolic status; the initial uptake is still controlled by GLTs, primarily ASCT2/SLC1A5, although it can also be transported by sodium-dependent neutral amino acid transporters (SLC38 family) LAT1 and LAT2, ATB0+/SLC6A14, and others.111 This may apply to most of the glutamate-based radiotracers (L-[5-11C]glutamine, 4-(2S,4R)4-fluoroglutamine), although perhaps not all, as [18F](2S,4S)-4-(3-fluoropropyl)glutamine appears more sensitive to the LAT1 transporter system.107,108

As increased proliferation is characteristic of tumors, markers of increased DNA synthesis have also been of significant interest for PET radiotracer development. The first such radiotracer, [11C]thymidine, was synthesized in 1972.112 Thymidine is transported into cells, likely by the equilibrative nucleoside transporter ENT1/SLC29A1, and then readily phosphorylated by thymidine kinase and incorporated into DNA. [11C]Thymidine has been extensively studied using PET imaging, but interpretation of in vivo pharmacokinetics as representing DNA synthesis is complicated by the extensive metabolism of [11C]thymidine to circulating radiolabeled metabolites. That difficulty prompted the synthesis of alternative nucleoside structures that are substrates for thymidine kinase, not incorporated into DNA, and resistant to metabolism: three ([11C]FMAU, [18F]FMAU, and [18F]FLT: Fig. 7) have been successful in human imaging studies.113 All of these require as an initial step the cellular uptake through the ENT/SLC29A1 transporter,114 just as for [11C]thymidine. Inside cells they are mono-phosphorylated and trapped, as there are no outwardly directed nucleotide transporters on the cell membranes. Thus, much like [18F]FDG, a radiotracer such as [18F]FLT is irreversibly trapped within the tumor cells through the combined actions of a transporter and an enzyme; differences in presence or function of the nucleoside transporter ENT1/ SLC29A1 can play an important role in the varying tumor localization of [18F]FLT and similar radiolabeled nucleosides. Another widely used approach to imaging proliferating tumors is the use of radiolabeled amino acids. Labeling amino acids with carbon-11 or fluorine-18 is, in fact, one of the earliest applications of PET radiochemistry, and a very long list of potential radiotracers have been radiolabeled with carbon11 or fluorine-18, including both natural and synthetic amino acids.115 Many if not most of these have been evaluated for tumor imaging in preclinical or clinical studies. For every radiolabeled amino acid, irrespective of whether it eventually is or is not incorporated into newly synthesized proteins, metabolized for energy, or simply trapped in the tissues, the initial step in uptake and localization is through transporters; furthermore, as noted earlier, there are 10 SLC families that can function to transport amino acids, suggesting that care should be taken to not ascribe tumor or tissue uptake to a single transporter. The L-type amino acid transporter 1 (LAT1/SLC7A5)

ARTICLE IN PRESS 12 is a transporter responsible for cellular uptake of the most widely used amino acid radiotracer, [11C]methionine, as well as a variety of fluorine-18 labeled aromatic amino acids. The LAT1 is also responsible for tumor uptake of L-6-[18F]fluoroDOPA, the amino acid developed originally for neurochemical imaging of the dopaminergic system in the brain, but of interest now for glioma imaging. A fluorine-18 labeled tryptophan derivative, L-1-[18F]fluoroethyl-tryptophan has been proposed as a radiotracer for tryptophan metabolism via indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway, of interest for assessment of cancer immunotherapy responses. The radiotracer localizes in tumors, but the uptake is a complex mixture of actions of transporters (eg, LAT1 and ASCT2/SLC1A5) and the enzymes involved in metabolism.116 As essentially all of the natural amino acids of interest have been radiolabeled, recent efforts in amino acid tracer development for tumor imaging have largely involved nonnatural amino acids and transporters other than the LAT1. The XC− system, which is actually a heterodimer of two transporters (SLC7A11 and SLC3A2), functions for the initial uptake of [18F]FSPG ((4S)-4-(3-[18F]fluoropropyl)-L-glutamic)117 and 5-[18F]fluoro-aminosuberic acid.118 The system A (alaninepreferring transporters, ASCT1/SLC1A4 and ASCT2/SLC1A5) is a transporter for important new radiotracers entering clinical studies, such as carbon-11 and fluorine-18 labeled derivatives of 2-amino-isobutyric acid (AIB),119 [18F]FACBC (fluciclovine), 120 4-[ 18 F]fluoroglutamine, 121 and the b 0,+ AT/SLC7A9 system a transport mechanism for a [18F]fluoroethylamino-tyrosine amino acid derivative. As an example of how transporters can have negative effects on radiotracer utility, one can consider the case of 3-[18F]fluoro-α-methyl-tyrosine: a radiolabeled amino acid developed for tumor imaging via uptake by the LAT1, its use in renal carcinoma is limited by high background due to uptake of the radiotracer by the organic ion transporters (eg, OAT1/SLC22A6). 122 The labeling of a dipeptide, [11C]glycine-sarcosine ([11C]GlySar), provided a tumor imaging agent based on the expression of the polypeptide transporter PEPT2/SLC15A2.123 Finally, radiolabeled choline analogs have been used to image the increased synthesis of phospholipids in tumors. Choline (2-hydroxy-N,N,N-trimethylethan-1-aminium) is a simple small molecule, but has been radiolabeled using both carbon-11 (isotopic substitution of one of the N-methyl groups) and fluorine-18 (substitution of a methyl group with a [18F]fluoroalkyl group).124,125 Both [11C]choline and the [18F]fluoromethyl derivative (termed [18F]fluorocholine) are in widespread use as tumor imaging agents, particularly for prostate cancer.126 As choline and fluorocholine are ionic quaternary amines, entry into tumor cells requires the action of transporters, with four types implicated in cancer: high-affinity choline transporters (CHT1/SLC5A7), intermediate-affinity choline-transporter-like proteins (CTLs/ SLC44A1-5), low-affinity organic cation transporters (OCTs/ SLC22A1-2), and organic cation/carnitine transporters (OCTNs),127 with most interest in the dual increase in both the CTLs and choline kinase activity in tumors.128 Thus,

M.R. Kilbourn [11C]choline and [18F]fluorocholine are radiotracers where the transporter function is required for successful in vivo imaging.

ABC Transporters and PET Imaging: Friend or Foe? The ABC transporters are the second large family of transporters found in the human body.8 Much as is found for the SLC transporters, there is an ABC transporter for anions, cations, amino acids, lipophilic molecules, short peptides, and even small proteins, and transporters are found on both cellular and intracellular membranes throughout the body. As the ABC transporters use ATP hydrolysis as an energy source, they are capable of transporting molecules against a concentration or ion gradient, and function to move molecules from the cytoplasm to outside the cell, or into intracellular organelles. These transporters play a crucial role in normal physiology, can be dysregulated in disease,129 and impact pharmacology due to the many ABC transporters that function as multidrug resistance transporters. This latter property also affects much of PET radiotracer development, and ABC extrusion transporters can be responsible for failures of imaging agents to localize in expected target organs, and the ability to predict such on the basis of chemical structure is still challenging. In comparison to the extensive number of PET radiotracers developed that involve the SLC transporters, efforts in the ABC transporter arena have been much more limited and more focused on three transporters: the p-glycoprotein Pgp/ ABCB1, multidrug resistance protein MRP/ABCC1, and the breast cancer resistance protein BCRP/ABCG2.130 Most studies have been of PET radiotracers and these proteins at the BBB, but applications of ABC transporter imaging in tumors have also been described: the action of these transporters can significantly interfere with therapeutic anticancer drugs. Most of the PET radiotracer development has been for imaging the P-glycoprotein (Pgp/ABCB1) presence or function. As advantages, Pgp is the most extensively characterized of the ABC transporters; many therapeutic drugs are substrates for Pgp, and there are multiple inhibitors known: there should thus be many choices for radiotracer selection. As disadvantages, uptake into a tissue (eg, brain) of a radiolabeled Pgp substrate is very limited (the transporter is actively effluxing the radiotracer), compounds often do not show selectivity over other ABC transporters, and Pgp inhibitors may also show properties of being substrates. Nevertheless, a number of radiolabeled compounds have been prepared and used to study Pgp in animals and humans. Verapamil (a calcium channel blocker) and loperamide (an opioid receptor agonist) are two examples of drugs developed for other purposes, but their high susceptibility to Pgp transport (and thus exclusion from the brain) made them candidates for radiolabeling for the purpose of imaging Pgp function in the BBB. Both have been prepared in carbon-11 labeled forms and as expected showed low uptake into the brains of animals and humans, but the uptake can be dosedependently increased by administration of a Pgp inhibitor such as cyclosporine A or taquidar. Higher brain uptake of

ARTICLE IN PRESS PET Tracers for Transporters both radiotracers could also be demonstrated in Pgp knockout mice. Attempts were then made to improve both radiotracers: a radiochemical synthesis was developed for the single resolved R-isomer of [ 11 C]verapamil, and [11C]N-desmethyl-loperamide (a metabolite of loperamide) was synthesized as a possibly more effective in vivo Pgp radiotracer. However, all of these radiotracers suffer from the in vivo formation of metabolites that are also Pgp-active. The limitations to use of [11C]verapamil and [11C]N-desmethylloperamide have however not discouraged investigators from using the radioligand in vivo in humans. A number of additional compounds expected to be Pgp substrates have been radiolabeled and tested as potential radiotracers, including [ 11 C]carvedilol, [ 11 C]phenytoin, [ 11 C]colchicine, [ 11 C]daunorubicin, [ 11 C]docetaxel, [11C]gefitinib, [18F]fluoropaclitaxel, and others.116 None of these have been as well studied as [ 11 C]verapamil or [11C]N-desmethyl-loperamide, although [11C]phenytoin has reached human studies.131 Thus, there remains a lack of an optimal Pgp substrate radiotracer. As an alternative to Pgp substrates, several Pgp inhibitors have been radiolabeled as potential radioligands for the protein. The lead compound was tariquidar, a Pgp inhibitor in clinical trials as adjunctive therapeutic drug in oncology. Initial studies used [11C]tariquidar, with subsequent efforts at improvement leading to a series of analogs ([11C]elacridar, [18F]fluoroethyl-elacridar, 1-[18F]elacridar, and [11C]laniquidar. [11C]Tariquidar and [11C]elacridar have seen utilization in human PET studies of the Pgp.130 Unfortunately, both radiotracers (as well as similar ones) have limitations. The selectivity of these radiotracers for Pgp over BCRP has been questioned, and when administered in the microdose amounts typical for a PET radiotracer, both radiotracers appear to exhibit properties of a Pgp substrate (brain uptake increased upon attempts to block Pgp with inhibitors). The search for a specific inhibitor radioligand for Pgp continues, and perhaps efforts to construct novel molecules such as [11C]MC18 may prove successful, although additional work is still needed to validate such new radiotracers.132 Radiotracer development for the breast cancer resistance protein (BCRP/ABCG2) has been very limited, largely due to the significant overlap in substrate specificity between the BCRP and Pgp. Nevertheless, a few compounds have been radiolabeled as potential in vivo BCRP radiotracers.130 Dantrolene is a muscle relaxant that was labeled with carbon-11 and nitrogen-13, but no in vivo biological data have yet been reported. [11C]SC-62807, a metabolite of the cyclooxygenase-2 inhibitor celecoxib, was evaluated as a substrate for the BCRP in mouse liver133: sensitivity to BCRP was demonstrated by using knockout mouse models, but further evaluation is still necessary, as in vitro the radiotracer is also sensitive to the organic anion SLC transporters OATP1B1 and OATP1B3. In an example completely unrelated to all of the prior structures of transporter ligands or substrates, [68Ga]galmydar, a lipophilic metalloprobe, was shown to exhibit BCRP and Pgpdependent in vivo uptake into the brain using knockout mice studies; the authors suggest galmydar could be a novel model for further radiotracer development.134

13

Figure 8 A radiolabeled prodrug approach to imaging the multidrug resistance protein (MRP1). Radiotracer (7-bromopurine) enters the brain through passive diffusion, reacts with glutathione, and the resulting polar metabolite extruded by the MRP1.

The final ABC transporters that have seen attention from radiopharmaceutical chemists are the multidrug resistance proteins MRP1/ABCC1 and MRP2/ABCC2.130 These have been of interest for brain imaging, as one of the three types of extrusion transporters present in the BBB, but also in other tissues such as the liver, where they are important for movement of conjugated molecules out of the hepatocytes and into the bile. As potential liver MRP2 substrates, a radiolabeled leukotriene (N-[11C]acetyl-LTE4) and the compound 15R-[11C]TIC have been tested in rats with positive early results, but again much more work needs to be done. For brain imaging of the MRP1, an interesting and novel approach has been proposed (Fig. 8) using radiolabeled 6-bromo-purines as prodrugs for formation of a molecule that would be efficiently transported out of the brain. The concept is that the radiotracer is lipophilic and enters the brain by passive diffusion, whereupon it is rapidly converted to a glutathione conjugate (conjugation via glutathione transferases is one mechanism used to remove toxic compounds from the brain) and the conjugates are efficiently transported out via the MRP1. Washout of brain radioactivity then reflects expulsion of the glutathione conjugates, and studies in knockout mice showed 90% slower efflux rate, consistent with an action of the MRP1. This concept has been demonstrated using both a carbon-11 and fluorine-18 labeled radiotracers.135 Issues with transporter specificity still need to be addressed, but the concept of combining prodrugs with efflux transporters is novel and intriguing.

The Future of Transporter Radiotracers The SLC and ABC transporters play a role in normal physiology, disease processes, and therapeutic drug action in tissues throughout the body. There may be proteins outside of the SLC and ABC families that possibly function as transporters: an example is the synaptic vesicle protein 2A, a puzzling protein whose function is not yet fully characterized136 but is for now assigned to the major facilitator superfamily (MFS) of transporters,5 and for which an exciting group of new radiotracers137-139 has been prepared as nonselective nerve

ARTICLE IN PRESS 14 terminal markers. The selectivity of transporters for a substrate can be exploited as part of an approach for reporter gene imaging: transfection of stem cells or tumor cells with the genes for metal ion transporters such as the copper transporter 1 (CTR1/SLC31A1) or the divalent metal ion transporter 1 (DMT1/SLC11A2) can be coupled with simple use of the appropriate positron-emitting metal radionuclide (64Cu and 52 Mn, respectively).140,141 Transporters are a very large group of proteins, and of the approximately 430 identified SLC transporters in the human body, as many as 30% remain orphan transporters, whose function and importance in human health and disease are yet to be elucidated.142 Future PET radiotracer development for transporters is thus very likely to continue.

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