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Fluorine-for-hydrogen: a strategy for radiolabeling, not a replacement
Nuclear Medicine and Biology 40 (2013) 956–958
Contents lists available at ScienceDirect
Nuclear Medicine and Biology journal homepage: www.elsevier.com/locate/nucmedbio
Editorial
Fluorine-for-hydrogen: a strategy for radiolabeling, not a replacement
1. Introduction As the development of new radiopharmaceuticals for in vivo imaging in Nuclear Medicine has moved towards the targeting of high affinity/low density sites on proteins, chemists have faced the challenges of incorporating appropriate radionuclides into molecules without compromising the in vivo biochemical properties such as affinities, specificities, metabolism and pharmacokinetics. The challenges of incorporation of technetium-99m, the workhorse radionuclide of clinical Nuclear Medicine, into small molecules have been recently reviewed [1]. With perhaps the exception of [ 99mTc]TRODAT1, a small molecule targeting the neuronal membrane dopamine transporter, success in this area has been very limited to relatively large molecules (peptides, antibody fragments) and contrasts with the greater success of radiolabeling small molecules with fluorine-18, a positron-emitting radionuclide with a much, much smaller impact on the overall molecular size of the radiopharmaceuticals. In a subsequent article, Duatti posited that the development of fluorine-18 labeled radiopharmaceuticals for PET has been based on “…a notable misconception propagated after the advent of the tracer 2-deoxy-2-[18F]fluoro-D-glucose”, using the “…widely accepted assumption that replacement of hydrogen with fluorine in a molecule has a minimal impact on its molecular properties simply because these two atoms behave almost identically as brightly demonstrated by the observed properties of [ 18F]FDG” [2]. As a radiopharmaceutical chemist long associated with the design and synthesis of fluorine-18 radiopharmaceuticals, I can assure the readers of Nuclear Medicine and Biology that this is far from the truth. The unique properties of fluorine have been long recognized in medicinal chemistry, so much so that incorporation of fluorine atoms into new drug molecules is a widespread occurrence. From a simple practical standpoint, fluorine substitution is most often for a hydrogen or a hydroxyl group, but the effect of such a substitution can vary tremendously, and not simply be that the fluorine atom “mimics” the replaced substituent; fluorine substituents, due to the stronger C–F bond, can block metabolic processes using hydroxylation of C–H bonds; substitution with fluorine, particularly for trifluoromethyl groups, can increase steric bulk relative to methyl groups; additions of fluorine atoms can either increase lipophilicity (as in the case of aromatic compounds) or decrease lipophilicity (as for aliphatic compounds); incorporation of fluorines can change the pKa values of carboxylic acids, alcohols and protonated amines; and substitution of fluorine for hydrogen can add a new site for hydrogen bonding. All of these are well characterized aspects of fluorine chemistry [3], well known before the advent of [ 18F]FDG. The development of fluorine-18 labeled radiopharmaceuticals has not been driven by a desire to copy a “successful” F-for-H substitution in [ 18F]FDG, but rather a desire to develop PET radiopharmaceuticals 0969-8051/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nucmedbio.2013.08.006
appropriate for more widespread use (one does not need an onsite cyclotron to utilize isotope, and products can be distributed), or to study biochemical processes that might be too slow for the 20-min half-life of carbon-11. Interestingly, although fluorine is present in a high fraction of clinically important drugs (e.g., Lipitor (atorvastatin) and Advair (containing fluticasone propionate)), most clinical drugs have not simply been suitable for development into imaging radiopharmaceuticals, most likely due to the fact that their selection and commercialization were based on ADME (absorption, distribution, metabolism and elimination) and not for the optimal target selectivity and pharmacokinetics sought in a radiotracer. Radiopharmaceutical chemists may not have deliberately proposed fluorine-for-hydrogen “substitutions”, but in practice have done exactly that. Has that been successful? It's not a simple question, as it depends on the criteria one uses to evaluate the radiotracer. It is likely that no such substitution of F-for-H has resulted in a molecule that in vivo has been an exact mimic of the parent molecule. There are, however, a number of examples other than [ 18F]FDG that can be examined to evaluate if fluorine-for-hydrogen substitutions have been successful, using the criteria that the fluorinated radiotracer should provide useful biochemical information (value) compared to the parent, hydrogen-containing radiotracer. 2. Target: High-affinity binding sites Much of radiopharmaceutical chemistry has been devoted to the design and synthesis of radiotracers that target high-affinity binding to proteins, such as receptors, transporters and enzymes. The examples below illustrate how fluorine substitution, intended to provide a means for introducing the radionuclide, can have varied effects. One of the earliest evaluations of the effects of fluorine substitution was series of N-alkyl and N-fluoroalkyl derivatives of spiperone, a high affinity ligand for the dopamine receptor in the brain [4,5]. As shown in Table 1, the introduction of a fluorine into the propyl chain of Npropylspiperone has different effects depending on the position of the fluorine substituent. When compared to the N-propyl analog, for the N-(2-[18F]fluoro)propylspiperone both in vitro binding affinity and lipophilicity were decreased; for the N-(3-[ 18F]fluoro)propyl analog, binding affinity was increased and lipophilicity even further decreased. The results of in vivo testing in the rat brain? The 3-[ 18F]fluoropropyl derivative exhibited the best specific binding (measured as the targetto-nontarget ratio, striatum/cerebellum), even better than the parent radiotracer, [ 18F]spiperone. This behavior was continued in studies through PET imaging in the monkey and human brain. There are other examples of fluorine-for-hydrogen substitutions in alkyl chains. In targeting the GABAA/benzodiazepine receptor complex, [ 18F]fluoroflumazenil (FFMZ) has been synthesized as an alternative to [ 11C]flumazenil. These two radiotracers are identical
Editorial / Nuclear Medicine and Biology 40 (2013) 956–958 Table 1 Relative binding affinities (RBA), lipophilicities, tissue concentrations and striatum/ cerebellum (S/C) ratios for spiperone and three N-alkyl and fluoroalkyl derivatives. Compound
RBA
Log P HPLC
%ID/g 2 min
%ID/g 60 min
S/C 60 min
Spiperone (SP) N-PropylSP N-2-18F-PropylSP N-3-18F-PropylSP
100 135 92 146
3.37 4.35 4.11 3.75
0.68 0.93 1.17 0.90
0.44 0.34 0.52 0.36
4.47 8.76 3.83 10.4
(0.06) (0.2) (0.16) (0.08)
(0.12) (0.25) (0.14) (0.05)
(0.14) (0.03) (0.04) (0.04)
(0.29) (0.76) (0.20) (1.53)
RBA — relative binding affinity (spiperone = 100), S/C = striatum/cerebellum. Values are mean (SD).
except for the addition of the fluorine atom at the end of the ethyl ester group. Adding the fluorine atom did not significantly affect the in vitro binding affinities (FMZ = 2.0 ± 0.9 nM, FFMZ = 1.45 ± 0.26 nM), but differences in the rate of metabolism and type of metabolite formed were possibilities. Significant and rapid metabolism of [ 18F]FFMZ to form lipophilic metabolites was observed in the rat [6], compromising the utility of [ 18F]FFMZ in that species, but such was not observed in the human plasma. [ 18F]FFMZ remains a viable choice for imaging the GABAA receptor in humans [7]. For the development of radiotracers for the muscarinic cholinergic receptor, Eckelman and coworkers directly compared [ 11C]propylTZTP and 3-[ 11C]fluoropropyl-TZTP as high affinity agonists selective for the M2 receptor subtype in the baboon brain [8]. For these compounds, the fluoride ion incorporation reduced the binding affinity (slightly) and lowers the log D; the brain pharmacokinetics of the fluoropropyl derivative were slightly faster, but the regional distribution volumes (estimates of specific binding) in baboon brain were not significantly different for the two radiotracers. To be fair, F-for-H substitution in alkyl chains does not always work. An excellent example is the attempt to add fluorine to the D2/3 agonist radiotracer PHNO, an N-propyl substituted naphthoxazine which has seen use in human studies in carbon-11 labeled form. The synthesis of an N-3-[ 18F]fluoropropyl derivative proved feasible, but despite an encouraging in vitro binding affinity for the D2 receptor (F-PHNO, Ki = 0.4 nM; PHNO, Ki = 0.6 nM), studies in rats showed a completely homogeneous brain distribution with no evidence of specific binding [9]. Fluorine substitution for hydrogen is not limited to short alkyl chains, but has been successfully accommodated in alkyl ring structures (e.g., 16-α-[ 18F]fluoroestradiol), and aryl rings. Examples of the latter include such pairs of radioligands as [ 18F]GBR13119 and [ 18F]GBR12909, and [ 11C]cocaine and 4-[ 18F]fluorococaine; in each pair, the difference between radiotracers is a single aryl fluorine substituent producing little differences of in vitro or in vivo characteristics. The targeted binding site for both sets was the neuronal membrane dopamine transporter (DAT); [ 18F]GBR12909 successfully localized in the striatum but proved in human studies to provide images of poorer quality than those obtained with the tropane-based DAT imaging agents [10], and was not further pursued. 4-[ 18F]Fluorococaine exhibited equivalent in vivo pharmacokinetics as compared in baboon brain to [ 11C]cocaine [11], making the F-for-H substitution successful although the very rapid brain pharmacokinetics of cocaine would not efficiently capitalize on the longer half-life of the fluorine radionuclide. What can we conclude from such examples as these? For binding studies, the F-for-H substitution is commonly attempted, and ofttimes successful. More challenging, perhaps, are radiotracers which function as substrates for active processes such as enzymes or transporters, as we shall see in the next section.
3. Metabolic radiotracers A second important type of PET radiotracer is those in which the molecule is designed to be a substrate for a particular transporter or
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enzyme (or combination of those). The in vivo image is a result of the final product or transported species being trapped in tissues, by perhaps an active or passive mechanism (e.g., transport into a vesicle, or cell membrane impermeability) or by incorporation into macromolecules (e.g, amino acids into proteins). The most obvious example of this is indeed [ 18F]FDG, the localization of which involves both transporter (the glucose transporter) and enzyme (hexokinase) actions. But it is not the only example. 6-[ 18F]FluoroDOPA is a widely used radiotracer that is a simple fluorinated derivative of the endogenous precursor to dopamine, 3,4dihydroxyphenylalanine (DOPA). In vivo, DOPA and 6-[ 18F]fluoroDOPA are substrates for the enzyme DOPA decarboxylase, with the products being dopamine and 6-[ 18F]fluorodopamine, respectively. Both of those amines are readily transported by the vesicular monoamine transporter type 2 (VMAT2) and accumulated into storage vesicles in monoaminergic neurons: the catecholamines are also subject to further conversion to 3-O-methyl derivatives by the enzyme catechol-O-methyl transferase. Is 6-[ 18F]fluoroDOPA a perfect mimic of DOPA? Comparisons of the two using double-label experiments [12] and syntheses of carbon-11 labeled forms of both [13] clearly show they are not equivalent, particularly in the rates of formation of the 3-O-methyl metabolite. It’s perfectly understandable: the fluorine substituent on the aryl ring undoubtedly influences the pKa of the catechol hydroxyls. It is perhaps not inconceivable that the fluorine substituent might also have minor effects on the decarboxylation step, on the rate of VMAT2 transport into the vesicle, and on the catabolism by monoamine oxidases. However, these slight differences have not compromised the effective use of 6-[ 18F] fluoroDOPA as a radiotracer in studies of human neurological diseases. A second example is [ 18F]fluoroacetate, proposed as a radiotracer that would substitute for [ 11C]acetate metabolism. Fluoroacetate follows the first initial steps of the citric acid cycle (TCA), as it is converted to fluoroacetyl-CoA and then to (2R,3R)-fluorocitric acid by the action of citrate synthase (acetate is converted to citric acid). Both citric acid and fluorocitric acid continue along the same steps, the next being dehydration by the actions of the enzyme aconitase, and it is at that step that the pathways diverge. Citric acid is then hydroxylated to give isocitric acid, and proceeds onwards in the TCA cycle: that is the fate of [ 11C]acetate. In contrast, fluorocitric acid undergoes hydroxylation-defluorination to yield (R)-hydroxy-transaconitic acid, a compound with high affinity for the aconitase enzyme and which effectively acts as a inhibitor of further TCA action, leading to the well known toxicity of fluoroacetic acid [3]. So much like [ 18F] FDG, [ 18F]fluoroacetic acid is a radiotracer that localizes due to a failure to progress along a known metabolic pathway. Is it a true mimic of acetate metabolism? No, it only provides information on the first steps of the TCA cycle, but still is an interesting radiotracer in human studies [14]. As a final example, we can consider the use of radiolabeled thymidines for measuring the rate of proliferation [15]. The synthesis of carbon-11 labeled thymidine actually predates that of [ 18F]FDG, with the preparation by Christman et al. in 1972 of [methyl- 11C] thymidine [16]. This was supplanted some years later by a more convenient synthesis of 2-[ 11C]thymidine. Thymidine is incorporated into DNA after the stepwise addition of three phosphate groups, beginning with phosphorylation by thymidine kinase. Interpretation of [ 11C]thymidine pharmacokinetics is complicated as the formation and kinetics of the metabolite [ 11C]CO2 need to be considered, but multi-compartmental analysis techniques have proven capable of providing good estimates of the rate of incorporation into DNA. To extend this technique to fluorine-18 labeling, several thymidine analogs and derivatives have been made, the most studied being 3′[ 18F]fluoro-3′-deoxythymidine (FLT). The fluorinated thymidine is a substrate for phosphorylation by the first enzyme, thymidine kinase (although at a slower rate than thymidine itself), but due to the lack of the 3′-hydroxyl group does not proceed with further phosphorylation
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Editorial / Nuclear Medicine and Biology 40 (2013) 956–958
steps. The tissue trapping is then of the monophosphorylated, charged species, in much the same fashion as for [ 18F]FDG. So is [ 18F]FLT a true mimic of thymidine? Strictly speaking, no: at best it is a measure of thymidine kinase activity, which may correlate with DNA synthesis. Thus, whereas [ 11C]thymidine ends up in DNA, phosphorylated [ 18F] FLT ends up trapped in the cytosol, and those are not equivalent end products. Nevertheless, use of [ 18F]FLT is widespread and has shown quite useful results in human studies in oncology.
assumption, as proposed by Duatti, that radiopharmaceutical chemists have considered fluorine as a simple nonisotopic substitution for hydrogen does not give the credit due to a generation of talented chemists who have carefully developed and validated a growing collection of highly useful fluorine-18 labeled radiopharmaceuticals for imaging of human physiology and disease. Any other conclusion is a misconception. Michael R. Kilbourn Division of Nuclear Medicine Department of Radiology University of Michigan Medical School Ann Arbor, MI 48109 E-mail address: [email protected]
4. Imaging agents or poisons? Finally, much of the article by Duatti is devoted to a discussion that PET radiotracers are actually poisons to biological systems. This is a disservice to the casual readership: any chemical is toxic in the wrong quantities or at the wrong location. The most toxic chemical in the world is water (think: drowning). Labeling radiotracers, as a class, as poisons is a misleading statement: numerous non-poisonous radiotracers come to mind, such as most radiolabeled compounds found as endogenous species in the mammalian body (e.g., radiolabeled acetate, palmitate, ammonia, methionine, choline), and even xenobiotics can have essentially no toxicity (e.g., N-methylpiperidinyl propionate, used in carbon-11 labeled form to study cerebral acetylcholinesterase). Duatti is correct that there are several examples of radiolabeled molecules that indeed would function as poisons, if administered at the wrong mass dose, but the wonderful advantage of the tracer methodology is that we can safely avail ourselves of these molecules for human imaging. 5. Conclusions So after reviewing these additional examples of F-for-H substitution (and there are dozens more possible), what might be the conclusion? Is it, in the words of Duatti regarding F-for-H substitution, “…their supposed chemical equivalence is one of the most popular assumptions granted by many radiochemists attempting to design tracers for positron emission tomography”? Is that true? The examples given here represent the efforts from some of the leading PET research groups over the last forty years. For radiotracers targeting high-affinity binding sites on proteins, F-for-H substitutions are quite often but not always well tolerated, and in a number of examples might actually improve the in vivo characteristics of the radiotracer. In contrast, the incorporation of fluorine atoms into compounds that undergo enzyme-mediated chemical changes can result in altered rates of reactions or the blocking of significant enzymatic steps. It is however doubtful that the design and syntheses of fluorine-18 radiotracers were naively done assuming fluorine can simply substitute for a hydrogen atom. Is that then a failure of the non-isotopic substitution? The extensive use of such radiotracers as 6-[ 18F]fluoroDOPA, [ 18F]FLT and many others certainly argues against that conclusion when the criterion for success or failure is whether the radiotracer provides useful biochemical information regarding physiology or disease. Fluorine is a highly useful substituent in medicinal chemistry: its importance can be seen in the high fraction of clinically-used drugs that contain it. Fluorine-18 is likewise a highly useful radionuclide, providing options for distribution and imaging that simply don't exist for carbon-11, or other very short half-life radionuclides. The
References [1] Eckelman WC, Jones AG, Duatti A and Reba RC. Progress using Tc-99 m radiopharmaceuticals for measuring high capacity sites and low density sites. Drug Discovery Today 2013 (in press). [2] Duatti A. Nonisotopic substitution: is fluorine a replacement for hydrogen? Nucl Med Biol 2013 (in press). [3] Yamazaki T, Taguchi T, Ojima I. Unique properties of fluorine and their relevance to medicinal chemistry and chemical biology. In: Ojima I, editor. Fluorine in medicinal chemistry and chemical biology. Wiley-Blackwell: United Kingdom; 2009. p. 3–46. [4] Welch MJ, Chi DY, Mathias CJ, et al. Biodistribution of N-alkyl and N-fluoroalkyl derivatives of spiroperidol: radiopharmaceuticals for PET studies of dopamine receptors. Nucl Med Biol 1986;13:523–6. [5] Welch MJ, Katzenellenbogen JA, Mathias CJ, et al. N-(3-[F-18]fluoropropyl)spiperone: the preferred F-18 labeled spiperone analog for positron emission tomographic studies of the dopamine receptor. Nucl Med Biol 1988;15: 83–97. [6] Dedeurwaerdere S, Gregoire M-C, Vivash L, et al. In-vivo imaging characteristics of two fluorinated flumazenil radiotracers in the rat. Eur J Nucl Med Mol Imaging 2009;36:958–65. [7] Park H-J, Kim CH, Park ES, et al. Increased GABA-A receptor binding and reduced connectivity at the motor cortex in children with hemiplegic cerebral palsy: a multimodal investigation using 18 F-fluoroflumazenil, immunohistochemistry, and MR imaging. J Nucl Med 2013;54:1263–9. [8] Reid AE, Ding Y-S, Eckelman WC, et al. Comparison of the pharmacokinetics of different analogs of 11C-labeled TZTP for imaging muscarinic M2 receptors with PET. Nucl Med Biol 2008;35:287–98. [9] Vasdev N, Seeman P, Garcia A, et al. Syntheses and in vitro evaluation of fluorinated nathoxazines as dopamine D2/D3 receptor agonists: radiosynthesis, ex vivo biodistribution and autoradiography of [18 F]F-PHNO. Nucl Med Biol 2007;34:195–203. [10] Koeppe RA, Kilbourn MR, Frey KA, et al. Imaging and kinetic modeling of [F-18] GBR 12909, a dopamine uptake inhibitor. J Nucl Med 1990;31:720. [11] Gatley SJ, Yu D-W, Fowler JS, et al. Studies with differentially labeled [11C]cocaine, [11C]norcocaine, [11C]benzoylecgonine, and [11C]- and 4’-[18 F]fluorococaine to probe the extent to which [11C]cocaine metabolites contribute to PET images of the baboon brain. J Neurochem 1994;62:1154–62. [12] Melega WP, Luxen A, Perlmutter MM, et al. Comparative in vivo metabolism of 6-[18F]fluoro-L-dopa and [3H]L-dopa in rats. Biochem Pharmacol 1990;39: 1853–60. [13] Hartvig P, Lindner KJ, Tedroff J, et al. Regional brain kinetics of 6-fluoro-(beta-11C)11 L-DOPA and (beta- C)-L-DOPA following COMT inhibition. A study in vivo using positron emission tomography. J Neural Transm Gen Sect 1992;87:15–22. [14] Ponde DE, Dence CS, Oyama N, et al. 18 F-Fluoroacetate: a potential acetate analog for prostate tumor imaging — in vivo evaluation of [18 F]fluoroacetate versus 11Cacetate. J Nucl Med 2007;48:420–8. [15] Krohn KA, Grierson JR, Muzi M, et al. Radiopharmaceuticals for imaging proliferation. In: Welch MJ, CaEckelman W, editors. Targeted molecular imaging. New York: CRC Press; 2012. p. 231–52. [16] Christman D, Crawford EJ, Friedkin M, et al. Detection of DNA synthesis in intact organisms with positron-emitting (methyl-11C)thymidine. Proc Natl Acad Sci USA 1972;69:a988–992.
Contents lists available at ScienceDirect
Nuclear Medicine and Biology journal homepage: www.elsevier.com/locate/nucmedbio
Editorial
Fluorine-for-hydrogen: a strategy for radiolabeling, not a replacement
1. Introduction As the development of new radiopharmaceuticals for in vivo imaging in Nuclear Medicine has moved towards the targeting of high affinity/low density sites on proteins, chemists have faced the challenges of incorporating appropriate radionuclides into molecules without compromising the in vivo biochemical properties such as affinities, specificities, metabolism and pharmacokinetics. The challenges of incorporation of technetium-99m, the workhorse radionuclide of clinical Nuclear Medicine, into small molecules have been recently reviewed [1]. With perhaps the exception of [ 99mTc]TRODAT1, a small molecule targeting the neuronal membrane dopamine transporter, success in this area has been very limited to relatively large molecules (peptides, antibody fragments) and contrasts with the greater success of radiolabeling small molecules with fluorine-18, a positron-emitting radionuclide with a much, much smaller impact on the overall molecular size of the radiopharmaceuticals. In a subsequent article, Duatti posited that the development of fluorine-18 labeled radiopharmaceuticals for PET has been based on “…a notable misconception propagated after the advent of the tracer 2-deoxy-2-[18F]fluoro-D-glucose”, using the “…widely accepted assumption that replacement of hydrogen with fluorine in a molecule has a minimal impact on its molecular properties simply because these two atoms behave almost identically as brightly demonstrated by the observed properties of [ 18F]FDG” [2]. As a radiopharmaceutical chemist long associated with the design and synthesis of fluorine-18 radiopharmaceuticals, I can assure the readers of Nuclear Medicine and Biology that this is far from the truth. The unique properties of fluorine have been long recognized in medicinal chemistry, so much so that incorporation of fluorine atoms into new drug molecules is a widespread occurrence. From a simple practical standpoint, fluorine substitution is most often for a hydrogen or a hydroxyl group, but the effect of such a substitution can vary tremendously, and not simply be that the fluorine atom “mimics” the replaced substituent; fluorine substituents, due to the stronger C–F bond, can block metabolic processes using hydroxylation of C–H bonds; substitution with fluorine, particularly for trifluoromethyl groups, can increase steric bulk relative to methyl groups; additions of fluorine atoms can either increase lipophilicity (as in the case of aromatic compounds) or decrease lipophilicity (as for aliphatic compounds); incorporation of fluorines can change the pKa values of carboxylic acids, alcohols and protonated amines; and substitution of fluorine for hydrogen can add a new site for hydrogen bonding. All of these are well characterized aspects of fluorine chemistry [3], well known before the advent of [ 18F]FDG. The development of fluorine-18 labeled radiopharmaceuticals has not been driven by a desire to copy a “successful” F-for-H substitution in [ 18F]FDG, but rather a desire to develop PET radiopharmaceuticals 0969-8051/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nucmedbio.2013.08.006
appropriate for more widespread use (one does not need an onsite cyclotron to utilize isotope, and products can be distributed), or to study biochemical processes that might be too slow for the 20-min half-life of carbon-11. Interestingly, although fluorine is present in a high fraction of clinically important drugs (e.g., Lipitor (atorvastatin) and Advair (containing fluticasone propionate)), most clinical drugs have not simply been suitable for development into imaging radiopharmaceuticals, most likely due to the fact that their selection and commercialization were based on ADME (absorption, distribution, metabolism and elimination) and not for the optimal target selectivity and pharmacokinetics sought in a radiotracer. Radiopharmaceutical chemists may not have deliberately proposed fluorine-for-hydrogen “substitutions”, but in practice have done exactly that. Has that been successful? It's not a simple question, as it depends on the criteria one uses to evaluate the radiotracer. It is likely that no such substitution of F-for-H has resulted in a molecule that in vivo has been an exact mimic of the parent molecule. There are, however, a number of examples other than [ 18F]FDG that can be examined to evaluate if fluorine-for-hydrogen substitutions have been successful, using the criteria that the fluorinated radiotracer should provide useful biochemical information (value) compared to the parent, hydrogen-containing radiotracer. 2. Target: High-affinity binding sites Much of radiopharmaceutical chemistry has been devoted to the design and synthesis of radiotracers that target high-affinity binding to proteins, such as receptors, transporters and enzymes. The examples below illustrate how fluorine substitution, intended to provide a means for introducing the radionuclide, can have varied effects. One of the earliest evaluations of the effects of fluorine substitution was series of N-alkyl and N-fluoroalkyl derivatives of spiperone, a high affinity ligand for the dopamine receptor in the brain [4,5]. As shown in Table 1, the introduction of a fluorine into the propyl chain of Npropylspiperone has different effects depending on the position of the fluorine substituent. When compared to the N-propyl analog, for the N-(2-[18F]fluoro)propylspiperone both in vitro binding affinity and lipophilicity were decreased; for the N-(3-[ 18F]fluoro)propyl analog, binding affinity was increased and lipophilicity even further decreased. The results of in vivo testing in the rat brain? The 3-[ 18F]fluoropropyl derivative exhibited the best specific binding (measured as the targetto-nontarget ratio, striatum/cerebellum), even better than the parent radiotracer, [ 18F]spiperone. This behavior was continued in studies through PET imaging in the monkey and human brain. There are other examples of fluorine-for-hydrogen substitutions in alkyl chains. In targeting the GABAA/benzodiazepine receptor complex, [ 18F]fluoroflumazenil (FFMZ) has been synthesized as an alternative to [ 11C]flumazenil. These two radiotracers are identical
Editorial / Nuclear Medicine and Biology 40 (2013) 956–958 Table 1 Relative binding affinities (RBA), lipophilicities, tissue concentrations and striatum/ cerebellum (S/C) ratios for spiperone and three N-alkyl and fluoroalkyl derivatives. Compound
RBA
Log P HPLC
%ID/g 2 min
%ID/g 60 min
S/C 60 min
Spiperone (SP) N-PropylSP N-2-18F-PropylSP N-3-18F-PropylSP
100 135 92 146
3.37 4.35 4.11 3.75
0.68 0.93 1.17 0.90
0.44 0.34 0.52 0.36
4.47 8.76 3.83 10.4
(0.06) (0.2) (0.16) (0.08)
(0.12) (0.25) (0.14) (0.05)
(0.14) (0.03) (0.04) (0.04)
(0.29) (0.76) (0.20) (1.53)
RBA — relative binding affinity (spiperone = 100), S/C = striatum/cerebellum. Values are mean (SD).
except for the addition of the fluorine atom at the end of the ethyl ester group. Adding the fluorine atom did not significantly affect the in vitro binding affinities (FMZ = 2.0 ± 0.9 nM, FFMZ = 1.45 ± 0.26 nM), but differences in the rate of metabolism and type of metabolite formed were possibilities. Significant and rapid metabolism of [ 18F]FFMZ to form lipophilic metabolites was observed in the rat [6], compromising the utility of [ 18F]FFMZ in that species, but such was not observed in the human plasma. [ 18F]FFMZ remains a viable choice for imaging the GABAA receptor in humans [7]. For the development of radiotracers for the muscarinic cholinergic receptor, Eckelman and coworkers directly compared [ 11C]propylTZTP and 3-[ 11C]fluoropropyl-TZTP as high affinity agonists selective for the M2 receptor subtype in the baboon brain [8]. For these compounds, the fluoride ion incorporation reduced the binding affinity (slightly) and lowers the log D; the brain pharmacokinetics of the fluoropropyl derivative were slightly faster, but the regional distribution volumes (estimates of specific binding) in baboon brain were not significantly different for the two radiotracers. To be fair, F-for-H substitution in alkyl chains does not always work. An excellent example is the attempt to add fluorine to the D2/3 agonist radiotracer PHNO, an N-propyl substituted naphthoxazine which has seen use in human studies in carbon-11 labeled form. The synthesis of an N-3-[ 18F]fluoropropyl derivative proved feasible, but despite an encouraging in vitro binding affinity for the D2 receptor (F-PHNO, Ki = 0.4 nM; PHNO, Ki = 0.6 nM), studies in rats showed a completely homogeneous brain distribution with no evidence of specific binding [9]. Fluorine substitution for hydrogen is not limited to short alkyl chains, but has been successfully accommodated in alkyl ring structures (e.g., 16-α-[ 18F]fluoroestradiol), and aryl rings. Examples of the latter include such pairs of radioligands as [ 18F]GBR13119 and [ 18F]GBR12909, and [ 11C]cocaine and 4-[ 18F]fluorococaine; in each pair, the difference between radiotracers is a single aryl fluorine substituent producing little differences of in vitro or in vivo characteristics. The targeted binding site for both sets was the neuronal membrane dopamine transporter (DAT); [ 18F]GBR12909 successfully localized in the striatum but proved in human studies to provide images of poorer quality than those obtained with the tropane-based DAT imaging agents [10], and was not further pursued. 4-[ 18F]Fluorococaine exhibited equivalent in vivo pharmacokinetics as compared in baboon brain to [ 11C]cocaine [11], making the F-for-H substitution successful although the very rapid brain pharmacokinetics of cocaine would not efficiently capitalize on the longer half-life of the fluorine radionuclide. What can we conclude from such examples as these? For binding studies, the F-for-H substitution is commonly attempted, and ofttimes successful. More challenging, perhaps, are radiotracers which function as substrates for active processes such as enzymes or transporters, as we shall see in the next section.
3. Metabolic radiotracers A second important type of PET radiotracer is those in which the molecule is designed to be a substrate for a particular transporter or
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enzyme (or combination of those). The in vivo image is a result of the final product or transported species being trapped in tissues, by perhaps an active or passive mechanism (e.g., transport into a vesicle, or cell membrane impermeability) or by incorporation into macromolecules (e.g, amino acids into proteins). The most obvious example of this is indeed [ 18F]FDG, the localization of which involves both transporter (the glucose transporter) and enzyme (hexokinase) actions. But it is not the only example. 6-[ 18F]FluoroDOPA is a widely used radiotracer that is a simple fluorinated derivative of the endogenous precursor to dopamine, 3,4dihydroxyphenylalanine (DOPA). In vivo, DOPA and 6-[ 18F]fluoroDOPA are substrates for the enzyme DOPA decarboxylase, with the products being dopamine and 6-[ 18F]fluorodopamine, respectively. Both of those amines are readily transported by the vesicular monoamine transporter type 2 (VMAT2) and accumulated into storage vesicles in monoaminergic neurons: the catecholamines are also subject to further conversion to 3-O-methyl derivatives by the enzyme catechol-O-methyl transferase. Is 6-[ 18F]fluoroDOPA a perfect mimic of DOPA? Comparisons of the two using double-label experiments [12] and syntheses of carbon-11 labeled forms of both [13] clearly show they are not equivalent, particularly in the rates of formation of the 3-O-methyl metabolite. It’s perfectly understandable: the fluorine substituent on the aryl ring undoubtedly influences the pKa of the catechol hydroxyls. It is perhaps not inconceivable that the fluorine substituent might also have minor effects on the decarboxylation step, on the rate of VMAT2 transport into the vesicle, and on the catabolism by monoamine oxidases. However, these slight differences have not compromised the effective use of 6-[ 18F] fluoroDOPA as a radiotracer in studies of human neurological diseases. A second example is [ 18F]fluoroacetate, proposed as a radiotracer that would substitute for [ 11C]acetate metabolism. Fluoroacetate follows the first initial steps of the citric acid cycle (TCA), as it is converted to fluoroacetyl-CoA and then to (2R,3R)-fluorocitric acid by the action of citrate synthase (acetate is converted to citric acid). Both citric acid and fluorocitric acid continue along the same steps, the next being dehydration by the actions of the enzyme aconitase, and it is at that step that the pathways diverge. Citric acid is then hydroxylated to give isocitric acid, and proceeds onwards in the TCA cycle: that is the fate of [ 11C]acetate. In contrast, fluorocitric acid undergoes hydroxylation-defluorination to yield (R)-hydroxy-transaconitic acid, a compound with high affinity for the aconitase enzyme and which effectively acts as a inhibitor of further TCA action, leading to the well known toxicity of fluoroacetic acid [3]. So much like [ 18F] FDG, [ 18F]fluoroacetic acid is a radiotracer that localizes due to a failure to progress along a known metabolic pathway. Is it a true mimic of acetate metabolism? No, it only provides information on the first steps of the TCA cycle, but still is an interesting radiotracer in human studies [14]. As a final example, we can consider the use of radiolabeled thymidines for measuring the rate of proliferation [15]. The synthesis of carbon-11 labeled thymidine actually predates that of [ 18F]FDG, with the preparation by Christman et al. in 1972 of [methyl- 11C] thymidine [16]. This was supplanted some years later by a more convenient synthesis of 2-[ 11C]thymidine. Thymidine is incorporated into DNA after the stepwise addition of three phosphate groups, beginning with phosphorylation by thymidine kinase. Interpretation of [ 11C]thymidine pharmacokinetics is complicated as the formation and kinetics of the metabolite [ 11C]CO2 need to be considered, but multi-compartmental analysis techniques have proven capable of providing good estimates of the rate of incorporation into DNA. To extend this technique to fluorine-18 labeling, several thymidine analogs and derivatives have been made, the most studied being 3′[ 18F]fluoro-3′-deoxythymidine (FLT). The fluorinated thymidine is a substrate for phosphorylation by the first enzyme, thymidine kinase (although at a slower rate than thymidine itself), but due to the lack of the 3′-hydroxyl group does not proceed with further phosphorylation
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steps. The tissue trapping is then of the monophosphorylated, charged species, in much the same fashion as for [ 18F]FDG. So is [ 18F]FLT a true mimic of thymidine? Strictly speaking, no: at best it is a measure of thymidine kinase activity, which may correlate with DNA synthesis. Thus, whereas [ 11C]thymidine ends up in DNA, phosphorylated [ 18F] FLT ends up trapped in the cytosol, and those are not equivalent end products. Nevertheless, use of [ 18F]FLT is widespread and has shown quite useful results in human studies in oncology.
assumption, as proposed by Duatti, that radiopharmaceutical chemists have considered fluorine as a simple nonisotopic substitution for hydrogen does not give the credit due to a generation of talented chemists who have carefully developed and validated a growing collection of highly useful fluorine-18 labeled radiopharmaceuticals for imaging of human physiology and disease. Any other conclusion is a misconception. Michael R. Kilbourn Division of Nuclear Medicine Department of Radiology University of Michigan Medical School Ann Arbor, MI 48109 E-mail address: [email protected]
4. Imaging agents or poisons? Finally, much of the article by Duatti is devoted to a discussion that PET radiotracers are actually poisons to biological systems. This is a disservice to the casual readership: any chemical is toxic in the wrong quantities or at the wrong location. The most toxic chemical in the world is water (think: drowning). Labeling radiotracers, as a class, as poisons is a misleading statement: numerous non-poisonous radiotracers come to mind, such as most radiolabeled compounds found as endogenous species in the mammalian body (e.g., radiolabeled acetate, palmitate, ammonia, methionine, choline), and even xenobiotics can have essentially no toxicity (e.g., N-methylpiperidinyl propionate, used in carbon-11 labeled form to study cerebral acetylcholinesterase). Duatti is correct that there are several examples of radiolabeled molecules that indeed would function as poisons, if administered at the wrong mass dose, but the wonderful advantage of the tracer methodology is that we can safely avail ourselves of these molecules for human imaging. 5. Conclusions So after reviewing these additional examples of F-for-H substitution (and there are dozens more possible), what might be the conclusion? Is it, in the words of Duatti regarding F-for-H substitution, “…their supposed chemical equivalence is one of the most popular assumptions granted by many radiochemists attempting to design tracers for positron emission tomography”? Is that true? The examples given here represent the efforts from some of the leading PET research groups over the last forty years. For radiotracers targeting high-affinity binding sites on proteins, F-for-H substitutions are quite often but not always well tolerated, and in a number of examples might actually improve the in vivo characteristics of the radiotracer. In contrast, the incorporation of fluorine atoms into compounds that undergo enzyme-mediated chemical changes can result in altered rates of reactions or the blocking of significant enzymatic steps. It is however doubtful that the design and syntheses of fluorine-18 radiotracers were naively done assuming fluorine can simply substitute for a hydrogen atom. Is that then a failure of the non-isotopic substitution? The extensive use of such radiotracers as 6-[ 18F]fluoroDOPA, [ 18F]FLT and many others certainly argues against that conclusion when the criterion for success or failure is whether the radiotracer provides useful biochemical information regarding physiology or disease. Fluorine is a highly useful substituent in medicinal chemistry: its importance can be seen in the high fraction of clinically-used drugs that contain it. Fluorine-18 is likewise a highly useful radionuclide, providing options for distribution and imaging that simply don't exist for carbon-11, or other very short half-life radionuclides. The
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