Given the increasing exploration of fluorescent tracers in the field of nuclear medicine, a need has risen for practical development guidelines that can help improve the translation aspects of fluorescent tracers. This editorial discusses the does and don’ts in developing fluorescence tracers. It has been put forward by the European Association of Nuclear Medicine (EANM) Translational Molecular Imaging &amp; Therapy committee and has been approved by the EANM board

Barbet, J

Caobelli, F

Cornelissen, B

de Jong, M

Del Vecchio,, S

Evangelista, L

Rbah-Vidal, L

van Leeuwen, F

Xavier, C

Given the increasing exploration of fluorescent tracers in the field of nuclear medicine, a need has risen for practical development guidelines that can help improve the translation aspects of fluorescent tracers. This editorial discusses the does and don’ts in developing fluorescence tracers. It has been put forward by the European Association of Nuclear Medicine (EANM) Translational Molecular Imaging & Therapy committee and has been approved by the EANM board

Cornelissen, Bart

Oxford University Research Archive

POSITION PAPER Open AccessGeneration of fluorescently labeled tracers– which features influence the translationalpotential?Fijs W. B. van Leeuwen1*, Bart Cornelissen2, Federico Caobelli3, Laura Evangelista4, Latifa Rbah-Vidal5,Silvana Del Vecchio6, Catarina Xavier7, Jacques Barbet5 and Marion de Jong8* Correspondence:F.W.B.van_Leeuwen@lumc.nl1Interventional Molecular ImagingLaboratory, Department ofRadiology, Leiden UniversityMedical Center, Leiden, theNetherlandsFull list of author information isavailable at the end of the articleAbstractGiven the increasing exploration of fluorescent tracers in the field of nuclear medicine,a need has risen for practical development guidelines that can help improve thetranslation aspects of fluorescent tracers. This editorial discusses the does and don’ts indeveloping fluorescence tracers. It has been put forward by the European Associationof Nuclear Medicine (EANM) Translational Molecular Imaging & Therapy committee andhas been approved by the EANM board.Keywords: Fluorescence, Image guided surgery, Molecular imaging, Tracers, Nuclearmedicine, Dual-modalityIntroductionWith the upcoming field of image-guided surgery, not only traditional 99mTc- and 111In-labelled radioguidance procedures blossom, but also the development of fluorescent andhybrid/bimodal tracers gains increasing interest. While converting efficient 68Ga-PETtracers for e.g. Prostate Specific Membrane Antigen to 99mTc- labeled versions for radio-guided surgery (Robu et al., 2017) is already part of the traditional radiochemistry skillset,attachment of fluorescent dyes as imaging labels requires additional expertise. Surpris-ingly, it is not fully recognized yet which modifications can be induced by the addition offluorescent dyes, e.g. alterations in biodistribution, and manuscripts seldomly report thekey analytical features required to objectively analyze them. In this editorial we aim todiscuss a number of factors to be taken into consideration when fluorescent dyes are usedin in vivo imaging tracers.Spectral propertiesFluorescence, a type of luminescence, is the result of the excitation of a conjugated systemfollowing the absorbance of light. When the excited electron reverts back to its groundstate it generates light with a longer wavelength (emission). In fluorescent organic dyes,electrons that can absorb high-energy photons are delocalized in connected p-orbitals dueto alternating single and double bonds (e.g. =C-C =C-). The absorbance spectrum (nm)and absorption maximum of dyes, and thus the in-depth excitability during an in vivo ap-plication (van Leeuwen et al., 2015), depends on the size and composition of the dye: theEJNMMI Radiopharmacy                and Chemistry © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 InternationalLicense (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, andindicate if changes were made.van Leeuwen et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:15 DOI 10.1186/s41181-017-0034-8bigger the conjugated system, the longer the wavelength it can absorb. This feature is wellillustrated by cyanine dyes Cy3, Cy5, and Cy7, which only differ in their bridge length, butdisplay an absorption maximum shift ranging from around 550 nm (visible) over 650 nm(far-red) to around 750 nm (near-infrared). Simply this means that a quest for near-infrared dyes converts to the use of large fluorescent labels and with that increases the riskthat the fluorescent label negatively influences the tracer pharmacokinetics. The efficiencyof a molecule absorbing light, the molar extinction coefficient (ε), also depends on thecomposition and surroundings of the dye, as does the conversion efficacy of the photonsabsorbed into emitted photons, the quantum yield (φ).Since the brightness of a fluorescent dye is proportional to the product of ε and φ, it iscompulsory that these should be accurately recorded and reported for each novel dye. Itshould be noted that these signature parameters could be considered as specific and criticalas the emission types and lifetimes of the radionuclides commonly applied in nuclear medi-cine. Contrary to radionuclides, the environment wherein the dyes reside strongly influ-ences their photophysical properties. Thus, researchers should realize that these propertiesmight vary when the dyes are conjugated to different vectors (e.g. peptide vs. protein) orwhen different pH values or solvents are used for analysis (e.g. serum vs. dimethylsufoxide(DMSO)). Hence, these features should ideally be recorded for each individual tracer andshould be measured in formulations that are representative for in vivo applications. Forexample, measuring the φ in DMSO could overestimate its value in an other environment).This is exemplified by indocyanine green, which has a φ of 12% in DMSO, yet 2% in salinebuffer (Benson & Kues, 1978).Unfortunately, for vectors with multiple conjugation sites, the quenching interac-tions between the dyes present on the same vector can influence the brightness of thetracer. Aggregating dyes residing on the same vector may quench each other’s fluores-cence, a feature most pronounced for slightly lipophilic and symmetrical dyes (vander Wal et al., 2016). It has even been shown that this effect can also occur betweendifferent types of dyes (Rood et al., 2014). Dyes that reside in 8–10 nm vicinity of eachother may display Förster resonance energy transfer, again limiting their fluorescenceintensity. Therefore, labeling ratios should be determined utilizing the Beer-Lambertlaw. Such analyses may, however, still be limited by the fact that stacking effects, ei-ther the result of non-covalent or covalent interactions, alter the absorbance profileof dyes. Stacking typically yields an additional lower absorbance peak in the case ofcyanine dyes (see Fig. 1). Hence, for an objective assessment of the number of dyeson e.g. a monoclonal antibody (mAb) it is key that authors, in addition to the abovefeatures, provide an absorbance spectrum of their compound in the formulation thatis applied to patients.Chemical radio- and photo-stabilityWhen administering tracers in vivo, stability becomes an issue, as metabolites may illumin-ate non-specific features. Enzymatic degradation of peptides is common (Nock et al.,2014), but peptidases are limited in their ability to metabolize synthetic dyes. Nevertheless,removal of the dye form the targeting vector should be deeply investigated. Degradation ofthe dye is most probable as the result of a nucleophilic attack on aryl ethers by primaryamines or thiols, which often occurs in an in vivo environment. Previously reported studiesvan Leeuwen et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:15 Page 2 of 6on these interactions indicate that they may induce dye disintegration and a reduction offluorescence intensity of e.g. ZW800-I (Hyun et al., 2014). Alternatively, thiol-based reac-tions with NIRDye 800 CW under the same conditions allow formation of entirely newmolecules (van der Wal et al., 2016). Both features are highly undesirable and should beevaluated and documented prior to using vectors labeled with dyes in vivo, particularly inhumans.As the intensity of the light emission is directly related to the absorption of light by afluorescent dye, manufacturers commonly enhance system performance by increasing theintensity of the excitation light source. Although this theoretically represents a validapproach, such modifications can also result in disintegration of the dye. This effect (photo-bleaching) is well known in molecular cell biology (Hoebe et al., 2007). As the bleaching be-havior is highly dye- and camera-dependent, its occurrence might prevent the surgical iden-tification of lesions under fluorescence guidance. Hence, the evaluation of photo-stability ofa fluorescent tracer in combination with the fluorescence camera intended for in vivo use isa key aspect in the translational process (van der Wal et al., 2016).A recent topic of interest has been the radio-stability of dyes in hybrid/bimodaltracers. It is not surprising that if dyes can be photo-bleached, irradiation, in particularβ- or α-emission, can induce radio-bleaching (Hernandez et al., 2017). This effect,which is radiation dose-dependent, is most likely to occur as result of prolonged expo-sures in the reaction mixture following radiolabeling, and does not occur when solu-tions of bimodal or hybrid tracers are used directly after production. To extend theshelf life of the reaction mixture, electron-scavengers can be applied to the formulation.As in vivo tumor accumulation commonly occurs at a relatively low concentration (<10% ID/g), radiobleaching is not likely to influence the in vivo use of such tracers.Pharmacokinetics and quantificationGiven that the fluorescent dyes used for in vivo imaging are relatively large molecules,it should come as no surprise that their conjugation may severely affect the affinity andFig. 1 Key features in the analysis of fluorescence tracers illustrated using (a) the conjugatable cyanine dyeCy5-sulfonate-COOH and the widely clinically applied dye Indocyanine green (ICG), b Absorbance and emis-sion spectrum of the non-conjugated dyes Cy5-sulfonate-COOH and ICG, c Changes in absorption spectrumas a result of conjugation, d Relevant photophysical parameters and their solvent dependencevan Leeuwen et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:15 Page 3 of 6in vivo kinetics of any targeting vector. Not only could the dye negatively influence re-ceptor interactions by inducing non-specific binding (Santini et al., 2016), it may alsoinfluence the cellular localization of some smaller vectors (Berkers et al., 2007). Whilesuch influences were expected for relatively small peptide-based vectors (Bunschoten etal., 2016), they have also been reported for much larger compounds, such as mAbs (Co-hen et al., 2011; Zhou et al., 2014). One key parameter in the in vivo pharmacokineticsis the dye’s interaction with serum proteins such as albumin. For ICG for example, thiseffect is so strong that it allows formation of non-covalent ICG-Albumin complexes(Bunschoten et al., 2012). Hence, it seems desirable to quantitatively document theseinteractions for new tracers.Fluorescence detection alone allows quantitative in vitro analysis, but quantitation ofin vivo effects are most practical for tracers that include a radiolabel. While critical forthe assessment of toxicity-related aspects, this information is generally missing in clin-ical reports on the use of fluorescent tracers (van Dam et al., 2011; Rosenthal et al.,2015; Burggraaf et al., 2015; Lamberts et al., 2017). Given the proven and well-acceptedpotential of radionuclide-based assessments by pharma and academia during drug andtracer development, it seems reasonable to advocate a quantitative pharmacokinetic as-sessment to become a standard requirement in reports on novel fluorescent tracers.Injected dose and cost impactBrightness, optical and in vivo stability, as well as pharmacokinetic properties combineddictate the tracer amount required for in vivo fluorescence imaging. In general, clinicaltrials use milligrams of fluorescent compounds, making them financially less achievablefor most hospitals. At the same time high doses may potentially lead to (partial) satur-ation of low capacity targets. Trials with hybrid tracers indicate that lower injecteddoses would still allow for fluorescence-based lesion identification (KleinJan et al.,2016). Importantly, the amount of tracer required for efficient imaging is directlyreflected in the cost of toxicity assessments and ultimately the cost of the tracer. Inaddition, the potential cost reduction to the overall healthcare system is critical for re-imbursement and wider clinical acceptance, and health economical assessments are anextremely useful inclusion when clinical trials with novel imaging agents are proposed.A relatively simple estimation of potential reimbursement cost for effective intraopera-tive visualization is the (% occurrence of failure) x (the average costs of follow up e.g.repeat surgery). For example, in expert surgical centers, breast cancer surgery is onlyirradical in 5% of the cases. Assuming that the cost of a repeat surgery is €5000 euro, re-imbursement of a tracer that allows for a theoretical 100% radical resection rate will theor-etically come to a value of €250 euro. Obviously this number is higher for surgicalprocedures with a higher failure rate and may increase if a clear improvement in the pa-tient’s quality of life can be realized. Nevertheless, cost efficiency should always be takeninto account when there are true translational aspirations for (fluorescent) tracers.ConclusionsFuture translation of fluorescent imaging agents strongly relies on more thoroughdocumentation of the compound’s photo-physical properties, chemo-, photo- andradio-stability, as well as translational aspects such as pharmacokinetics, dose and cost.van Leeuwen et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:15 Page 4 of 6AcknowledgementsThe authors wish to acknoweledge the EANM board for their official support for the editorial.FundingNot applicableAvailability of data and materialsNot applicableAuthors’ contributionsThe members of the EANM Translational Molecular Imaging & Therapy committee have jointly drafted the editorial.All authors read and approved the final manuscript.Ethics approval and consent to participateNot applicableConsent for publicationAll authors gave their consent for publication.Competing interestsNot applicablePublisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Author details1Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden University Medical Center, Leiden, theNetherlands. 2Department of Oncology, CRUK&MRC Oxford Institute for Radiation Oncology, University of Oxford,Oxford, UK. 3Department of Nuclear Medicine, University Hospital Basel, Basel, Switzerland. 4Nuclear Medicine andMolecular Imaging Unit, Veneto Institute of Oncology IOV – IRCCS, Padua, Italy. 5CRCINA, INSERM, CNRS, Universitéd’Angers, Université de Nantes, Nantes, France. 6Department of Advanced Biomedical Sciences, University Federico II,Naples, Italy. 7In vivo Cellular and Molecular Imaging Lab (ICMI)-Department, Vrije Universiteit Brussel, Ixelles, Belgium.8Department of Radiology & Nuclear Medicine, Erasmus Medical Centre, Rotterdam, the Netherlands.Received: 23 November 2017 Accepted: 23 November 2017ReferencesBenson RC, Kues HA. 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Generation of fluorescently labeled tracers – which features influence the translational potential?

Generation of fluorescently labeled tracers – which features influence the translational potential?

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