Protonation-Guided Design and Evaluation of Selective PET Tracers for Light Chain Cardiac Amyloidosis

Protonation-Guided Design and Evaluation of Selective PET Tracers for Light Chain Cardiac Amyloidosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Protonation-Guided Design and Evaluation of Selective PET Tracers for Light Chain Cardiac Amyloidosis Mengchao Cui, JiangXue Tang, Miao Wang, Yuying Li, Haonan Yu, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7823866/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Early detection of cardiac amyloidosis (CA) is challenging, despite advances in repurposed β -amyloid PET tracers and amyloid-targeted [ 124 I]Evuzamitide. The heterogeneity of CA subtypes requires invasive tests, like tissue biopsy, before targeted therapy can begin. Amyloidogenic light chains (AL) expose negatively charged pockets enriched in acidic residues and N-glycosylated modifications, guiding the design of selective molecular probes. We showed that protonation-driven recognition accomplishes unprecedented selectivity for AL deposits. Using this principle, we developed a fluorinated derivative, [ 18 F] FT-8 , based on a 4-pyridylpiperazine scaffold, which displayed high binding affinity ( K i = 11.52 nM) and selectivity towards AL, as well as favorable pharmacokinetics. In first-in-human PET studies, [ 18 F] FT-8 provided high-contrast visualization of AL deposits in the myocardium and extracardiac organs with significant specificity. These findings position [ 18 F] FT-8 as a promising PET tracer for advancing the non-invasive differential diagnosis of AL-CA and highlighting the role of protonation in developing new-generation molecular probes across diverse disease contexts. Biological sciences/Biological techniques/Imaging/Positron-emission tomography Health sciences/Cardiology/Cardiovascular biology/Cardiovascular diseases/Cardiomyopathies/Cardiac hypertrophy Health sciences/Diseases/Cardiovascular diseases/Cardiomyopathies/Cardiac hypertrophy Biological sciences/Biochemistry/Protein folding/Protein aggregation Health sciences/Biomarkers/Diagnostic markers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cardiac amyloidosis (CA) is a progressive infiltrative cardiomyopathy caused by the deposition of amyloid fibrils in the myocardial extracellular matrix, predominantly arising from light chain amyloidosis (AL) or transthyretin amyloidosis (ATTR). 1 – 3 AL-CA accounts for over half of all CA cases and stems from misfolded immunoglobulin light chains secreted by abnormal clonal plasma cell proliferation. 1 , 2 Among CA subtypes, AL-CA carries the worst prognosis, with median survival as short as six months, tenfold shorter than that of ATTR-CA. 2,4 Early diagnosis and precise subtyping are essential for guiding treatment, as each subtype follows a distinct clinical course and requires tailored therapies. However, clinical heterogeneity frequently leads to diagnostic delays. Once considered rare and usually diagnosed only post-mortem, CA is now increasingly recognized due to heightened awareness and advanced imaging technologies. Autopsy studies have reported CA in up to 43% of individuals aged ≥ 75 years, underscoring its underdiagnosis. 5 Current diagnostic guidelines (Fig. 1 a) recommend a multimodal approach that integrates electrocardiogram (ECG), echocardiography (Echo), cardiac magnetic resonance (CMR), laboratory testing (e.g., serum/urine analyses and TTR genotyping), endomyocardial biopsy (EMB), and nuclear imaging with single-photon emission computed tomography (SPECT) or positron emission tomography (PET). 2 – 4 While EMB with Congo red staining, immunohistochemistry, and mass spectrometry (MS)-based proteomics remains the diagnostic gold standard, it is invasive, carries procedural risks, and is subject to sampling error (potentially yielding false negatives). 6 Non-invasive approaches, although safer, often lack sensitivity, specificity, or the ability to quantify amyloid burden and subtype. Nuclear molecular imaging has transformed the field by enabling non-invasive and longitudinal visualization of disease-associated biomarkers. A major breakthrough was the clinical adoption of bone-avid SPECT tracers such as [ 99m Tc]pyrophosphate ([ 99m Tc]PYP), [ 99m Tc]3,3-diphosphono-1,2-propanodicarboxylic acid ([ 99m Tc]DPD), and [ 99m Tc]hydroxymethylene diphosphonate ([ 99m Tc]HMDP), which are now recommended as non-biopsy diagnostic tools for ATTR-CA. 7–9 However, [ 99m Tc]DPD also demonstrates myocardial uptake in ~ 40% of AL-CA cases and in rare forms of Apolipoprotein AI amyloidosis, complicating subtype differentiation. 10–12 PET tracers originally developed for β -amyloid (A β ) imaging in Alzheimer’s disease (AD), including [ 11 C]PiB, [ 18 F]Florbetaben, [ 18 F]Florbetapir, [ 18 F]Flutemetamol, and [ 18 F]Florbetazine (Fig. 1 b), have also shown affinity for both AL and ATTR fibrils, with higher sensitivity for AL. 13–16 The only tracer specifically designed for pan -amyloid imaging in CA to date is [ 124 I]Evuzamitide, a 45-amino acid peptide carrying 12 net positive charges (Fig. 1 c). This agent binds strongly through electrostatic interactions with anionic heparan sulfate proteoglycans (HSPGs), which are colocalized with amyloid deposits and are central to plaque formation. 17 – 22 Despite its broad amyloid-binding profile, [ 124 I]Evuzamitide lacks subtype specificity, necessitating confirmatory laboratory tests for AL-CA. Thus, there remains a pressing need for subtype-specific tracers, particularly for AL-CA, to enable early diagnosis, quantitative assessment, and treatment monitoring. To address this challenge, we focused on the distinct molecular features of amyloid fibrils. Compared with TTR, immunoglobulin light chains are more acidic and frequently N-glycosylated, generating localized negative charges along the β -strand axis. 23 – 27 These unique features provide opportunities for selective targeting with small-molecule cations. We hypothesize that positively charged molecules could first localize to acidic residues and N-glycosylation sites through electrostatic attraction, and subsequently engage hydrophobic β -sheet cavities via combined electrostatic, hydrophobic, hydrogen-bonding, and π-π stacking interactions. This mechanism differs from [ 124 I]Evuzamitide, which primarily targets HSPGs. Supporting this model, our previously reported tracer [ 18 F] 23 selectively bound AL deposits (Fig. 1 d), validating the complementary roles of electrostatic surface interactions and conformational β -sheet cavity recognition. A major limitation of permanently cationic compounds is their high baseline cardiac uptake, which results from pronounced mitochondrial trapping in cardiomyocytes. For instance, [ 18 F] 23 (log D 7.4 = 0.22) showed undesired retention in healthy myocardium. 15 Enhancing hydrophilicity or employing a dynamic protonation strategy could reduce off-target accumulation while preserving affinity. 28 To this end, we selected piperazine, a privileged N-heterocyclic scaffold widely used in drug development, for its synthetic versatility and tunable protonation states. 29 Pyridyl substituents incorporated into the diarylpiperazine scaffold exhibit position-dependent p K a variation, allowing fine-tuning of molecular charge to meet the desired physicochemical and biological properties. The piperazine ring also provides dual hydrogen-bonding with AL fibrils, while appended aromatic rings further contribute hydrophobic and π-π stacking interactions with β -sheets (Fig. 1 e). To test this dynamic protonation design, we synthesized 4-pyridyl, 2-pyridyl, and phenyl analogues representing high-, low-, and non-protonated forms. Their selectivity was systematically evaluated using autoradiography (ARG) on human myocardial tissues from autopsies and biopsies. The highly protonated 4-pyridylpiperazine scaffold was strategically diversified into nine fluorinated derivatives, which were comprehensively characterized through in vitro binding assays, mechanistic investigations, in vivo PET pharmacokinetics in rodents, and first-in-human PET imaging studies. Results and discussion N-Protonation governs selective recognition of AL deposits Given the advantageous properties of 125 I-labeled probes, including their long half-life (T 1/2 = 59.4 d) and high achievable molar activity ( A m = 81.4 GBq/μmol), which allow reliable detection of probe-target interactions in pathological specimens at extremely low concentrations by in vitro ARG. Guided by this rationale, we initially designed and synthesized three iodinated compounds ( 7 - 9 ) with distinct N-protonation states under physiological conditions (pH = 7.4). As outlined in Supplementary Scheme 1, the diarylpiperazine derivatives were effectively constructed in a one-step reaction via either Buchwald-Hartwig cross-coupling or ethylene glycol-mediated arylamination between amine and halides. The synthetic route involved an initial preparation of brominated intermediates, followed by Stille cross-coupling and subsequent iodination to produce the cold compounds 7 - 9 , with overall yields of 8 - 29% and purities exceeding 95% (Supplementary Fig. 1 and Supplementary Table 2). Measured p K a values revealed that the nitrogen atoms directly attached to the iodine-substituted aromatic rings remained unprotonated at physiological pH (7.4), as reflected by uniformly low p K a 1 values of 3.69, 3.54, and 3.98 (Fig. 2a). Instead, protonation occurred predominantly on the nitrogen atom located on the opposite aromatic ring. Among these compounds, the 4-pyridyl analogue 7 displayed the highest basicity (p K a 2 = 8.97), resulting in near-complete protonation (97.40%). In contrast, the 2-pyridyl analogue 8 showed only marginal protonation (2.03%), whereas the phenyl analogue 9 remained entirely unprotonated. Thus, these three compounds served as representative prototypes of high-, low-, and non-protonated small molecules, distinguished by the position or absence of a nitrogen atom. Radiolabeling of [ 125 I] 7-9 was achieved by iododestannylation with radiochemical yields (RCYs) of 66-83% and radiochemical purities (RCPs) over 95%, as verified by co-injection with corresponding cold compounds (Supplementary Fig. 2 and Supplementary Table 3). ARG on postmortem human myocardial slices was then conducted to evaluate their binding characteristics. [ 125 I] 7 demonstrated strong and selective binding in AL-positive samples, with only background signals from ATTR-positive and control tissues. By contrast, [ 125 I] 9 failed to differentiate between AL and ATTR deposits, resembling the behavior of established A β -PET tracers (Fig. 2a). [ 125 I] 8 exhibited similar labeling of both AL and ATTR pathologies, consistent with its low degree of N-protonation. Semi-quantitative analysis of the autoradiographic images further revealed a protonation-dependent selectivity, with tracer preference for AL over ATTR deposits positively correlating with the extent of protonation across the diarylpiperazine series (Fig. 2b). Notably, [ 125 I] 7 displayed a 5-fold selectivity for AL relative to ATTR, whereas [ 125 I] 8 yielded a near-equivalent selectivity ratio (1.20), and the non-protonatable [ 125 I] 9 exhibited the lowest ratio of 0.65. These findings provide initial validation for a protonation-guided tracer design strategy for selective targeting of cardiac AL deposits. Binding characteristics of hyper-protonated forms To elucidate the mechanism underlying the selective recognition of myocardial amyloid by these diarylpiperazine tracers, we next systematically characterized probe-target interactions. Given that protonated microspecies may preferentially associate with AL fibrils through electrostatic attraction, competitive binding assays were performed under increased ionic strength to disturb electrostatic contributions. In AL-rich regions, [ 125 I] 7 binding was markedly reduced, with a 94.67% ± 0.18% decrease in the presence of 1.0 M NaCl (Figs. 2c-2d and Supplementary Fig. 4a), whereas no ionic strength-dependent effects were observed for [ 125 I] 8 or [ 125 I] 9 . Likewise, the binding of [ 125 I] 8 to ATTR deposits remained unaffected by ionic strength variations (Figs. 2g-2h and Supplementary Figs. 7-8), indicating that electrostatic interaction is the dominant determinant of selectively targeting AL deposits. Hydrophobic interactions represent another major driving force in amyloid recognition. An 8-hour formic acid pretreatment, known to disrupt β -sheet structures, abolished the specific [ 125 I] 7 signals on AL-rich myocardial slices (Figs. 2e-2f). Compound 7 at 500 nM achieved 65.65% ± 1.10% self-inhibition, and as anticipated, formic acid treatment reduced [ 125 I] 7 binding to background levels (77.34% ± 2.80% reduction). This result provides direct evidence that hydrophobic interactions with β -sheet cavities are indispensable for AL recognition. On AL-enriched slices, co-incubation with 500 nM of unlabeled compounds 7 - 9 effectively inhibited [ 125 I] 8 binding by 78.50% ± 2.66%, 90.97% ± 0.28%, and 86.91% ± 0.13%, respectively (Fig. 2i and Supplementary Fig. 7). A similar inhibition profile was also observed for [ 125 I] 9 (Supplementary Fig. 8), indicating co-occupancy of compounds 7 - 9 within the β -sheet cavity of AL fibrils. At the same concentration, established A β -PET tracers (Florbetazine, PiB, and Florbetapir) also inhibited [ 125 I] 8 and [ 125 I] 9 binding with an average efficiency of 70.31% ± 6.80% and 74.23% ± 4.87%. Notably, the diarylpiperazine compounds 7 - 9 exhibited significantly stronger inhibition than these planar, conjugated A β tracers, suggesting that the piperazine linker may enhance binding within the AL fibril pocket. In contrast, competition assays on ATTR-rich slices revealed markedly different inhibition patterns. At 500 nM, compound 7 failed to displace either [ 125 I] 8 or [ 125 I] 9 from ATTR deposits (Fig. 2j and Supplementary Fig. 8). In comparison, compounds 8 and 9 at the same concentration reduced tracer binding by more than 50%. These findings, consistent with ARG results, suggest that while the diarylpiperazine scaffold adopts a conformation compatible with ATTR recognition, the high-protonated 4-pyridyl substituent in compound 7 may hinder such interactions owing to repulsion from the positively charged basic residues of TTR. Furthermore, 500 nM of Florbetazine inhibited ATTR binding of [ 125 I] 8 and [ 125 I] 9 by 58.77% ± 1.18% and 34.82% ± 0.77%, respectively, whereas PiB and Florbetapir produced minimal inhibition, in line with previous reports. 16,31 These results further reinforce the notion that the diarylpiperazine scaffold provides substantial potential for hydrophobic interactions within β -sheet cavities. Together, these experimental results provide preliminary support for our hypothesis that AL aggregates are more negatively charged along the β -strand axis than ATTR aggregates, likely reflecting a higher prevalence of acidic residues and N-glycosylation modifications. Such electrostatic landscapes promote the enrichment of β -sheet-recognizing molecules bearing positive charges, whether permanently cationic or protonatable, around AL deposits, followed by hydrophobic engagement with AL fibrils. Of particular note, the binding mechanism of the N-protonated tracer [ 125 I] 7 differs from that of the cationic polypeptide tracer [ 124 I]Evuzamitide described in the introduction, even though the latter also displayed pronounced inhibition (~ 90%) under high ionic strength (1.0 M NaCl). 32 To further validate the favorable properties of [ 125 I] 7 across the heterogeneity of light-chain sequences in pathological settings, additional myocardial specimens enriched with pathological confirmed AL or ATTR deposits by anti-lambda light chain antibody or anti-transthyretin antibody were examined by in vitro ARG (specimen details in Supplementary Table 1). 26,33 [ 125 I] 7 exhibited diffuse or focally increased uptake across multiple AL cases, including three autopsy and three biopsy specimens. Regions labeled by [ 125 I] 7 showed excellent co-localization with fluorescence staining (FS) using the reference tracer 23 (Fig. 3). By contrast, [ 125 I] 7 produced only uniform, weak signals in ATTR-rich slices, a pattern distinct from the profiles revealed by anti-transthyretin IF (Fig. 4). These findings consistently demonstrated the high selectivity and sensitivity of [ 125 I] 7 for AL over ATTR and non-amyloid controls. Quantitative self-displacement studies were then performed on adjacent myocardial slices using [ 125 I] 7 as the radioligand. Analysis of radioactivity density (Digital Light Units/mm², DLU/mm²) within AL-rich regions generated a displacement curve that yielded an IC 50 value of 10.62 ± 3.22 nM, indicating high affinity for AL deposits (Supplementary Fig. 5). Conversion into 18 F-labeled AL-PET tracers To extend the application of PET imaging for the selective diagnosis of AL-CA, we adopted a dual structural modification strategy to generate fluorinated 4-pyridylpiperazine derivatives as candidate AL-targeted tracers (Fig. 5a). Direct replacement of the iodine atom in lead compound 7 with fluorine yielded analogue FT-1 , which retained high affinity for native AL deposits in competitive binding assays with [ 125 I] 7 (IC 50 = 12.87 ± 2.54 nM; Fig. 5b and Supplementary Fig. 6). However, attempts to radiolabel FT-1 via its aryl-BPin precursor using Cu(py) 4 (OTf) 2 catalysis in TBAB/DMA/n-BuOH at 110 °C gave a RCY of only 0.6%, precluding further evaluation. A benzene analogue ( FT-2 ) also required aryl-BPin precursor, rendering it similarly suboptimal for large-scale preparation. We therefore pursued an alternative 18 F-labeling approach using trimethylammonium trifluoroacetate salts as precursors, which are more compatible with mild conditions. Through Buchwald-Hartwig arylamination, we synthesized FT-3 and FT-4 in 22% and 43% yield, respectively. Both compounds showed moderately reduced affinities (IC 50 = 64.27 ± 21.55 nM and 240.40 ± 67.01 nM, respectively). Notably, the para -fluorinated analogue FT-1 exhibited higher affinity than its meta -fluorinated counterpart FT-4 , suggesting a preference for the para -substitution site in AL binding. Despite the moderate affinities, in vitro ARG confirmed the selective binding of both tracers to AL deposits (Supplementary Fig. 11a). Importantly, [ 18 F] FT-3 and [ 18 F] FT-4 were radiolabeled with substantially improved RCYs (9%-11%, non-decay-corrected) using Kryptofix 222 /K 2 CO 3 in anhydrous acetonitrile at 80 °C. We next introduced 18 F at terminal alkyl positions via nucleophilic aliphatic substitution, an efficient route for radiolabeling. Through tailored synthetic methods, several side chains, including 2-fluoroethanol, chiral 3-fluoropropan-2-ol, and chiral 4-fluorobutane-2,3-diol, were incorporated into the aromatic system to yield analogues FT-5 to FT-9 (Supplementary Scheme 2). 34 In line with structure-activity relationship (SAR) trends for A β tracers, 2-fluoroethoxy substitution preserved favorable AL binding, whereas additional hydroxyl groups in ( S )-3-fluoro-2-hydroxypropanoxy and ((2 R ,3 S )-4-fluoro-2,3-dihydroxybutanoxy substituents weakened this interaction (Fig. 5b). 35 As anticipated, the meta -substituted 2-fluoroethoxy analogue FT-9 exhibited the lowest affinity (IC 50 > 1000 nM), whereas the para -substituted FT-5 and FT-8 displayed acceptable potency (IC 50 = 68.38 ± 14.95 nM and 11.61 ± 1.32 nM, respectively). Their tosylated precursors were efficiently radiofluorinated with RCYs up to 33% (non-decay corrected, Supplementary Scheme 3). All 18 F-labeled tracers showed RCP >95% after HPLC purification and were verified by co-injection with corresponding 19 F standards (Supplementary Fig. 3 and Supplementary Table 3), supporting their suitability for further bio-evaluation. Micro-PET pharmacokinetics in rats Next, [ 18 F] FT-3 -[ 18 F] FT-5 , and [ 18 F] FT-8 were prioritized for in vivo pharmacokinetic evaluation in rodents. Following intravenous injection ( i.v. ), all tracers rapidly entered systemic circulation, with immediate cardiac passage and the emergence of clear myocardial contours within one minute post-injection. The mean standard uptake value (SUV mean ) reached a peak of 5.03, 5.91, 4.94, and 5.60 for [ 18 F] FT-3 -[ 18 F] FT-5 and [ 18 F] FT-8 , respectively (Fig. 5c). Pulmonary uptake was observed in parallel, with [ 18 F] FT-4 showing the highest transient accumulation (SUV peak = 7.41) before redistribution (Fig. 5g and Supplementary Figs. 9d-9f). Subsequently, all tracers underwent rapid myocardial and pulmonary clearance, approaching background levels at 50-60 min, indicative of negligible non-specific retention in healthy tissues. Notably, [ 18 F] FT-8 displayed a delayed myocardial clearance during the first 10 minutes, but the SUV ratio (SUVR) between myocardium and anatomically adjacent tissues, particularly lung and blood pool, approached close 1.0 at 50-60 min, satisfying the requirements for cardiac imaging (Fig. 5d). Dynamic PET and maximum intensity projection (MIP, 60-70 min post-injection) analyses of these tracers revealed predominant hepatobiliary clearance (Figs. 5e-5f and Supplementary Figs. 9a-9c), in line with other lipophilic tracers. Only [ 18 F] FT-4 showed transient hepatic accumulation (SUV mean = 6.15 at 14-15 min post-injection), followed by partial clearance (SUV mean = 4.72 at 50-60 min), slightly below [ 18 F] FT-8 (SUV mean = 4.87 at 50-60 min, Supplementary Fig. 10). However, mild defluorination signals were observed in joints and thoracic vertebrae for [ 18 F] FT-4 . Overall, all radiolabeled candidates demonstrated favorable myocardial pharmacokinetics, but [ 18 F] FT-8 emerged as the most suitable lead, characterized by rapid myocardial uptake (SUV peak = 5.60), efficient washout (washout 1 min/60 min = 8.26), minimal residual activity in the blood-pool (SUV mean = 0.53 at 50-60 min) and lungs (SUV mean = 0.85 at 50-60 min), and the strongest AL-binding potency. Physiological hepatic and renal uptake did not interfere with cardiac visualization, consistent with previously reported small-molecule CA tracers. 4 For subsequent clinical translation, large-scale automated radiosynthesis is indispensable. As summarized in Fig. 6d, automated synthesis of [ 18 F] FT-8 achieved a non-decay corrected conversion yield of 30% and a molar activity of 159.62 GBq/μmol. Additionally, [ 18 F] FT-8 exhibited excellent stability in both saline and rat plasma, maintaining integrity (> 98%) for at least 8 hours in saline and 5 hours in rat plasma at 37 °C (Fig. 6c). No bone uptake was detected in living animals, further confirming its in vivo stability. These profiles collectively support the feasibility of advancing [ 18 F] FT-8 into clinical studies. UV-metric titration determined the p K a values of the aromatic nitrogen atoms in [ 18 F] FT-8 to be 2.97 and 8.76, corresponding to a dynamic protonation fraction of 95.85% under physiological conditions. Although the measured log D 7.4 was 1.11 ± 0.10, in vivo studies in rodents indicated that the equilibrium between protonated and deprotonated states minimizes undesirable electrostatic interactions with negatively charged intracellular components. This balance enhances membrane permeability and enables [ 18 F] FT-8 to selectively engage AL deposits in myocardial tissue without binding to mitochondria in healthy myocardium. Binding of [ 18 F]FT-8 to human AL pathologies The binding selectivity of [ 18 F] FT-8 was validated by in vitro ARG studies on an expanded cohort of myocardial samples, including six from patients with AL-CA, six with ATTR-CA, and two healthy controls. As shown in Fig. 6a, [ 18 F] FT-8 produced high-intensity signals across all AL samples, with distribution patterns consistent with staining by an anti-lambda light-chain antibody and the cationic tracer 23 . In contrast, only background-level signals were detected on slices from ATTR and healthy hearts. Saturation binding assays further confirmed the high affinity of [ 18 F] FT-8 for AL deposits, yielding a K d of 114.81 nM in case 5-6 (Fig. 6b). Moreover, a bone marrow specimen from a patient diagnosed with plasmacytoma and light chain restriction, a condition pathologically distinct but closely related to AL, also showed strong and specific [ 18 F] FT-8 binding (Supplementary Fig. 11b). These findings highlight the potential diagnostic utility of [ 18 F] FT-8 not only in cardiac amyloidosis but also in extracardiac light chain associated pathologies. In the presence of 500 nM FT-8 , [ 18 F] FT-8 binding was reduced by 80.56% ± 1.62% in AL-enriched regions, whereas only background-level signals persisted in areas of extensive transthyretin deposits (Figs. 7a, 7c, and Supplementary Fig. 4b). These self-block assays confirm that [ 18 F] FT-8 binds selectively and specifically to AL deposits. When challenged with increasing ionic strength, [ 18 F] FT-8 exhibited marked sensitivity, with AL bound radioactivity declining sharply to 21.88% ± 3.17% at 0.1 M NaCl and stabilizing at higher concentrations (Fig. 7d). By contrast, [ 18 F]Florbetazine binding was unaffected by NaCl concentrations up to 5 M, similar to [ 125 I] 8 - 9 , and the elevated signals observed in myocardial slices were attributable to salt precipitation (Fig. 7b). This differential behavior underscores the dominant contribution of the protonated microspecies (95.85%) of [ 18 F] FT-8 , which confers strong electrostatic avidity for AL binding. Consistent with expected hydrophobic interactions with β -sheet structures, A β -PET agents (PiB, Florbetapir, and Florbetaben) produced an average of 24.55% inhibition of [ 18 F] FT-8 to AL binding. Notably, when [ 18 F]Florbetazine was used as the radioligand, unlabeled FT-8 suppressed AL binding by 59.24% ± 1.60%, within the same range as inhibition by cold Florbetazine (48.13% ± 4.89%), while showing no blocking effect against ATTR deposits (Fig. 7e). These results suggest that FT-8 engages strong hydrophobic interactions on par with reported tracers, but such contacts represent secondary, weaker forces in ATTR deposits. The dominant mechanism of AL recognition by [ 18 F] FT-8 remains electrostatic avidity, which cannot be inhibited by unprotonated A β -PET tracers. First-in-human PET study of [ 18 F]FT-8 Encouraged by the preclinical data demonstrating the high affinity, selectivity, and favorable kinetics of [ 18 F] FT-8 in rodents, we performed an exploratory first-in-human PET study to characterize its pharmacokinetics and evaluate its potential to visualize AL deposits in a patient with systemic amyloidosis. [ 18 F] FT-8 was well tolerated, with no adverse events reported following tracer administration in either subject. In the healthy volunteer, [ 18 F] FT-8 rapidly accumulated in the myocardium, peaking at an SUV mean of 7.04 within 10 minutes post-injection (Figs. 8a-8b). Uptake was also observed in the intestine, kidneys, spleen, liver, lungs, brain, salivary glands, thyroid, and parotid gland. With the exception of the liver and intestine, tracer activity in all organs declined over time, parallel to blood clearance, generally consistent with rodent kinetics. As shown in Fig. 8b, myocardial uptake decreased by 45% within the first hour, reaching an SUV mean of 2.40 at 3 h post-injection (clearance ratio 2.94). Spleen and lung activity similarly diminished, yielding weak signals after 3 hours (SUV mean = 1.78 and 1.00, respectively). By contrast, persistent liver and intestinal uptake precluded imaging of these regions. Notably, the skeletal system showed only background activity, with no evidence of in vivo defluorination. Collectively, these results identify 3 h post-injection as an optimal imaging window, offering a favorable background for target visualization. On the basis of this biodistribution profile in healthy volunteer, we assessed organ-specific AL binding in a patient with systemic amyloidosis using PET/CT acquired at 3 h post-injection (Fig. 8c). [ 18 F] FT-8 showed pronounced and heterogeneous uptake in AL-burdened organs, including the left and right ventricular walls (SUV mean = 7.44), lungs (SUV mean = 6.77), spleen (SUV mean = 6.63), rectum (SUV mean = 4.63), and tongue (SUV mean = 2.82), clearly distinguishing the patient from the healthy control. A rectal biopsy specimen confirmed amyloid deposits by Congo Red staining under cross-polarized light and FS using the cationic tracer 23 (Fig. 8d). Specifically, IF analysis further identified the deposits as lambda light chain-derived, with positive anti-lambda and negative anti-transthyretin antibody signals. The concordance between in vivo uptake and ex vivo pathology supports [ 18 F] FT-8 as a specific AL tracer. Although bone-avid tracers have utility in ATTR amyloidosis, their mechanism of selectivity, binding to microcalcifications, can lead to false positives in AL cases. 36 Indeed, about 40% of AL patients in a cohort of 292 exhibited [ 99m Tc]DPD uptake in the myocardium, underscoring the risk of misclassification. 11 Current diagnostic pathways for ATTR require prior exclusion of AL, yet no selective radiotracers exist for AL itself, necessitating invasive biopsy. The ability of [ 18 F] FT-8 to identify AL deposits non-invasively thus represents a major advance, with potential to enable accurate differentiation of CA subtypes and to reduce reliance on tissue sampling. Collectively, these first-in-human results demonstrate that [ 18 F] FT-8 imaging can provide a safe, non-invasive means to detect both cardiac and extracardiac AL involvement, warranting further validation in larger cohorts and in ATTR populations. Outlook Given the aggressive progression of AL-CA, precise subtyping following early diagnosis is crucial for achieving the best outcomes. Future clinical practice is likely to demand diagnostic approaches that are simpler, more accurate, and non-invasive, with selective PET tracers emerging as powerful enablers. Distinct from conventional screening pipelines based on synthetic fibrils or animal models, the use of native amyloid deposits from patient myocardium is expected to preserve authentic conformations and post-translational modifications. This approach provides a clinically relevant substrate that can accelerate probe discovery, improve reliability, and validate translational potential. Protonation-driven recognition is emerging as a rational design principle with broad translational potential. At the tissue-level, hyper-protonated microspecies selectively bind AL deposits through electrostatic interactions with negatively charged amino acid residues and glycosylated motifs on fibril surfaces, reinforced by hydrophobic and hydrogen-bonding contacts with β -sheet structures. The optimized tracer, [ 18 F] FT-8 , exhibits predominant affinity, selectivity, and favorable kinetics, and can be readily synthesized in hospital settings. A single PET scan at 3 h post-injection achieves clear visualization of AL deposits without evidence of in vivo defluorination. Compared with [ 124 I]Evuzamitide, which suffers from long half-life, deiodination with free iodine accumulation in the thyroid (requiring potassium iodide prophylaxis), and delayed imaging kinetics, [ 18 F] FT-8 offers lower radiation exposure, simplified workflows, and a cleaner thyroidal background, enabling detection of rare thyroid involvement. Because AL amyloidosis frequently involves multiple extracardiac organs, this new tracer further enables simultaneous assessment of cardiac and systemic disease within a single scan. Its safety, dosimetry, sensitivity, and specificity are now being evaluated in larger clinical cohorts. Challenges remain in interpreting signals from the liver and intestine, which reflect high uptake and warrant further tracer optimization. Nonetheless, the framework established here provides a foundation for the development of new-generation tracers with preserved affinity and improved pharmacokinetics, and mature radiotracers could ultimately reshape the diagnostic pathway for CA. More broadly, the protonation-guided design strategy may extend beyond AL. ATTR protein, which is less acidic, may be more amenable to neutral or anionic ligands, whereas hyperphosphorylated tau protein in the brain may preferentially engage protonated tracers. Thus, protonation-sensitive probe design offers a generalizable framework for targeting proteins with defined charge-distribution patterns across a spectrum of diseases. Methods Chemistry and radiochemistry Synthetic procedures, purity analyses, and characterization data are provided in the Supplementary Information. Animals All procedures involving animals were approved by the Animal Care Committee of Beijing Normal University (Approval No. BNUCC-EAW-2023-0615-01). Human participants The first-in-human PET/CT study was approved by the Ethics Committee of Tianjin Medical University General Hospital (Approval No. IRB2023-YX-239-01) and registered at ClinicalTrials.gov (NCT07232459). Written informed consent was obtained from the participants or their caregivers. This study included one healthy volunteer (41 years old, 82 kg, male) and one patient diagnosed with systemic amyloidosis (71 years old, 53 kg, male). In vitro ARG Formalin-fixed paraffin-embedded myocardial slices were deparaffinized in xylene (10 min) and rinsed in EtOH. Pre-treated slices were incubated with either 125 I-labeled tracers (74 kMq/mL, 2 h) or 18 F-labeled tracers (0.74 MBq/mL, 1 h) at room temperature. Unbound tracer was removed by a rapid ethanol rinse and 50% ethanol washing (5 min). Slices were dried and exposed to a phosphor screen (10 h for 125 I-labeled tracers; 1 h for 18 F-labeled tracers). ARG images were acquired on a Cyclone Plus storage phosphor system (PerkinElmer, USA) and quantified using OptiQuant (PerkinElmer, USA) and ImageJ. Selectivity for AL versus ATTR aggregates was calculated as the following formula, $$\:\text{S}\text{e}\text{l}\text{e}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}\:=\frac{{AL}^{+}}{{ATTR}^{+}}$$ Where \(\:{AL}^{+}\) / \(\:{ATTR}^{+}\) was the main grey value with AL/ATTR pathology in red/blue circles (indicated in Fig. 2 a) calculated by ImageJ. Binding potency to AL deposits IC 50 and K d values were measured from ARG studies of serial myocardial slices from an AL-CA case. For IC 50 , pre-treated slices were incubated with a mixture of [ 125 I] 7 (74 kBq/mL) and increasing concentrations of unlabeled competitor (10 − 5 − 10 − 11 M) for 2 h at room temperature. For K d , pre-treated slices were incubated with a solution of gradient concentration of [ 18 F] FT-8 (0.23, 2.32, 11.6, 23.2, 58.0, 115.9, 231.8, 463.6 nM) for 1 h at room temperature. The washing, ARG image acquisition, and analysis were performed as described above in the in vitro ARG. Inhibition assays The inhibition ratio was measured from ARG studies of serial myocardial slices. Pretreated slices were incubated with solutions of [ 125 I] 7 – 9 (74 kBq/mL), [ 18 F] FT-8 (0.74 MBq/mL), or [ 18 F]Florbetazine (0.56 MBq/mL) in the presence of different concentrations of competing compounds (2 h for 125 I-labeled tracers; 1 h for 18 F-labeled tracers) at room temperature. The washing, ARG image acquisition, and analysis were performed as described above in the in vitro ARG. Distribution coefficient determination The logD 7.4 of [ 18 F ]FT-8 was determined in an n-octanol/PBS system. Briefly, [ 18 F] FT-8 (100 µL) was mixed with 3 mL 1× PBS and 2.9 mL n-octanol, vortexed (3 min), and centrifuged (3000 rpm, 10 min). The n-octanol phase was serially partitioned against fresh PBS. Aliquots (100 µL) from each phase were measured with a WALLAC/Wizard 2480 γ-counter (PerkinElmer, USA). Protonation state determination The p K a values were determined using UV spectroscopic titration on a Sirius T3 instrument (Sirius Analytical Instruments, UK) and analyzed with Sirius T3Dt software (v1.0.12.120) by WuXi AppTec Co., Ltd. (Shanghai, China). Fluorescence staining Pre-treated slices were incubated with compound 23 (1 µM, 10% ethanol) for 5 min at room temperature, rinsed in ethanol, and washed with water (1 min). Fluorescence images were captured on a DMi8 automated microscope imaging system (Leica, Germany) equipped with a Leica K8 (A23B726014) camera and appropriate filter sets. Immunofluorescence Antigen retrieval was performed in preheated buffers (lambda light chain: 0.01 M sodium citrate, pH = 6.0; transthyretin: Tris-EDTA, pH = 9.0) at 95°C for 15 minutes. Slices were blocked (3% BSA, 2 h) and incubated overnight at 4°C with primary antibody (anti-lambda light chain antibody, ab124719, 1:400 in TBS or anti-transthyretin antibody, 11891-1-AP, 1:100 in TBS). After TBST washes, slices were incubated with secondary antibody (goat anti-rabbit 488 nm, ab150077) at room temperature for 2 h. Nuclei were counterstained with DAPI. Fluorescence images were acquired on a DMi8 automated microscope imaging system (Leica, Germany). Pseudocolors were assigned to lambda light chain (yellow) and transthyretin (green). Congo Red staining Pre-treated slices were stained with Congo Red solution (#G1531, Solarbio) for 30 min at room temperature and imaged under polarized light using a DMi8 automated microscope imaging system (Leica, Germany) equipped with a FLEXACAM C3 (1923260073) camera. Micro-PET/CT imaging in rats. Male SD rats (6–10 weeks, male) were anesthetized with a mixture of isoflurane and air (2.5 L/min), and tail intravenously administered [ 18 F] FT-3 (7.205 MBq, n = 1, 10% ethanol), [ 18 F] FT-4 (7.293 MBq, n = 1, 10% ethanol), [ 18 F] FT-5 (6.013–6.839 MBq, n = 2, 10% ethanol) and [ 18 F] FT-8 (7.918–9.387 MBq, n = 2, 10% ethanol). PET/CT included dynamic cardiac imaging (0–60 min), whole-body PET scanning (60–70 min), and CT acquisition. Cardiac PET frames: 60 s × 15, 300 s × 5, 600 s × 2. Data were reconstructed by 3D-OSEM algorithm and analyzed by PMOD 4.0 software (PMOD Technologies, Switzerland). PET/CT imaging in humans One healthy volunteer (41 years old, 82 kg, male) and one patient with systemic amyloidosis (71 years old, 53 kg, male) underwent [ 18 F] FT-8 PET/CT imaging, using a Discovery 710 PET/CT system (GE Healthcare, USA) from the top of the head to mid-thigh. No fasting was required before the scanning. For healthy volunteer, following a low-dose, non-contrast CT acquisition (3.75 mm slice thickness, tube voltage: 120 kV, Scan Type: Helical, Rotation time: 0.8, Rotation Length: Full, Pitch & Speed: 1.375:1 & 55.00), [ 18 F] FT-8 (381.1 MBq) was administered intravenously at the bedside. Dynamic PET acquisition commenced immediately and continued for 6 consecutive whole-body scans (1 min/bed; 7 beds), followed by a single whole-body scan (2 min/bed; 7 beds). A second dynamic PET acquisition was performed at 120 min post-injection, consisting of 4 consecutive whole-body scans (2 min/bed; 7 beds). For the patient, PET scan was performed after a same low-dose, non-contrast CT acquisition from the top of the head to mid-thigh at 180 min post-injection (432.9 MBq). All PET data were reconstructed using ordered-subset expectation maximization (2 iterations, 8 subsets) with a Gaussian filter (full width at half maximum 5 mm, image size 168 × 168). All reconstructed images were surveyed in axial, coronal, and sagittal planes and analyzed by PMOD 4.1 (PMOD Technologies, Switzerland). Declarations Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability All supporting materials are available in the Supplementary Information. Source data are provided with this paper. The raw and analyzed datasets generated during this study are accessible for research purposes from the corresponding author upon reasonable request. Competing interests All authors declare no competing financial interests. Author contributions M.C. conceptualized the project and designed the methodology. M.C., Y.L., S.Y., J.D., L.Y., D.C., X.Y., and J.Z. contributed materials and funding. J.T., M.W., Q.Z., J.W., and Y.L. conducted all the preclinical experiments. J.T., S.Y., and X.Z. performed radiosynthesis. J.T., M.P., H.Y., and S.Y. carried out first-in-human PET imaging and data analysis. J.T. prepared the original draft, and Y.L., M.C., M.P., H.Y., and S.Y. reviewed and edited the manuscript. All authors approved the final version. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant Nos. 22376016, 22306015, 22022601, and U1967221). References Wechalekar, A. D., Gillmore, J. D. & Hawkins, P. N. Systemic amyloidosis. Lancet 387 , 2641–2654 (2016). Falk, R. H., Alexander, K. M., Liao, R. & Dorbala, S. AL (light-chain) cardiac amyloidosis. J. Am. Coll. Cardiol. 68 , 1323–1341 (2016). Ruberg, F. L., Grogan, M., Hanna, M., Kelly, J. W. & Maurer, M. S. Transthyretin amyloid cardiomyopathy. J. Am. Coll. Cardiol. 73 , 2872–2891 (2019). Fontana, M. et al. The last decade in cardiac amyloidosis. JACC Cardiovasc. Imaging 18 , 478–499 (2025). Porcari, A. et al. Incidence and characterization of concealed cardiac amyloidosis among unselected elderly patients undergoing post-mortem examination. Front. Cardiovasc. Med. 8 , 749523 (2021). Karameh, M. et al. Endomyocardial biopsy in clinical practice: The diagnostic yield and insights from a 5-year single-center experience. 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Varying levels of small microcalcifications and macrophages in ATTR and AL cardiac amyloidosis: Implications for utilizing nuclear medicine studies to subtype amyloidosis. Cardiovasc. Pathol. 25 , 413–417 (2016). Additional Declarations There is NO Competing Interest. Supplementary Files StudyprotocolforFT8.docx Clinical trial protocol NCT07130565.csv Clinical Trial Summary SupplementaryInformation.docx Supplementary Information Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7823866","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":536405562,"identity":"a758e2d9-6b48-4570-938b-d091ed107c73","order_by":0,"name":"Mengchao 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The cardiac SPECT image using [\u003csup\u003e99m\u003c/sup\u003eTc]PYP is reproduced from ref. 30. \u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e, Chemical structures of reported PET tracers for CA. The \u003cem\u003epan\u003c/em\u003e-amyloid tracer [\u003csup\u003e124\u003c/sup\u003eI]Evuzamitide is schematically illustrated, highlighting key positively charged lysine residues and the \u003csup\u003e124\u003c/sup\u003eI-labeling site. \u003cstrong\u003ee\u003c/strong\u003e, Chemical structure of the 4-pyridylpiperazine derivatives developed in this study, with predicted interactions with AL amyloid fibrils (λ isotype, PDB: 7NSL), including electrostatic, hydrophobic, and hydrogen-bonding contacts with negatively charged residues, glycosylation sites, or hydrophobic cavities. The fibril surface is color-coded by electrostatic potential (red, negative; blue, positive; white, neutral), adapted from ref. 26. Created in BioRender. Li, Y. (2025) https://BioRender.com/h85zcsu\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7823866/v1/53964cc74fe0188742618dc1.png"},{"id":101880951,"identity":"0457ec5f-aebf-4a20-8bec-7c7072eae8f0","added_by":"auto","created_at":"2026-02-04 15:08:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1944328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAffinity and selectivity of \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e125\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eI-labeled 4-pyridylpiperazine derivatives for AL deposits.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, ARG images of myocardial slices from a healthy control (HC, #Case 1), a patient with AL-CA (#Case 5-1), and a patient with ATTR-CA (#Case 10-1) incubated with [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e-\u003cstrong\u003e9\u003c/strong\u003e. The basic p\u003cem\u003eK\u003c/em\u003ea values of the nitrogen atoms and the corresponding N-protonation fractions of [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e-\u003cstrong\u003e9\u003c/strong\u003e at physiological pH (7.4) were experimentally measured. \u003cstrong\u003eb\u003c/strong\u003e, Semi-quantitative analysis of AL selectivity of [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7-9\u003c/strong\u003e based on ARG results. Regions of interest (ROIs) corresponding to AL and ATTR deposits are indicated by red and blue circles, respectively, in panel \u003cstrong\u003ea\u003c/strong\u003e. \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e, ARG images and binding percentage of [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e to AL-rich myocardial slices (#Case 5-2) under increasing ionic strength conditions and quantification from the red outlines in panel \u003cstrong\u003ec\u003c/strong\u003e. \u003cstrong\u003ee\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e, ARG images and binding percentage of [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e to AL-rich myocardial slices (#Case 5-6) in the presence of 500 nM of cold compound \u003cstrong\u003e7\u003c/strong\u003e or 8-hour pretreatment of formic acid quantified from the red circles in panel \u003cstrong\u003ee\u003c/strong\u003e. \u003cstrong\u003eg\u003c/strong\u003e-\u003cstrong\u003eh\u003c/strong\u003e, ARG images and binding percentage of [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e8\u003c/strong\u003e to AL-rich myocardial slices (#Case 5-7) under increasing ionic strength conditions quantified by red outlines in panel \u003cstrong\u003eg\u003c/strong\u003e. \u003cstrong\u003ei\u003c/strong\u003e-\u003cstrong\u003ej\u003c/strong\u003e, Inhibition assays conducted in the absence (control) or presence of 500 nM of cold compounds \u003cstrong\u003e7\u003c/strong\u003e-\u003cstrong\u003e9\u003c/strong\u003e, Florbetazine, PiB, or Florbetapir, on AL-rich\u003cem\u003e \u003c/em\u003e(#Case 5-7) and ATTR-rich (#Case 10-3) myocardial slices using [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e8\u003c/strong\u003e as the radioligand. Data bars represent mean ± s.d. (n = 3). Scale bars, 5 mm.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7823866/v1/0728b33a998b205beb10721d.png"},{"id":101881138,"identity":"c01bae0c-0a73-4896-8189-eec8bb8ff49f","added_by":"auto","created_at":"2026-02-04 15:10:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4966556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eARG images of multiple AL myocardial slices incubated with [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e125\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eI]7.\u003c/strong\u003e Fluorescence staining (FS) with the permanent cationic tracer \u003cstrong\u003e23\u003c/strong\u003e (red). Adjacent tissue slices from #Case 5-4 and #Case 5-5 were subjected to immunofluorescence staining (IF) with an anti-lambda light chain antibody (ab124719, yellow) and nuclear counterstaining with DAPI (blue). Scale bars, 2 mm (full-slice views) and 100 μm (magnified regions corresponding to solid and dashed boxes).\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7823866/v1/2407eb5aa123c0ff33823c19.png"},{"id":101880881,"identity":"4e666596-56d2-49f7-8e01-78ffb63e0556","added_by":"auto","created_at":"2026-02-04 15:07:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3670314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eARG images of multiple ATTR myocardial slices incubated with [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e125\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eI]7. \u003c/strong\u003eIF was conducted with an anti-transthyretin antibody (11891-1-AP, green) and nuclear counterstaining with DAPI (blue). Scale bars, 2 mm (ARG and full IF images) and 100 μm (magnified regions corresponding to solid and dashed boxes).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7823866/v1/414e20a3bf8f8feeaaa3da26.png"},{"id":101881102,"identity":"d99baa8a-24d5-4ca3-859d-e2a12659325a","added_by":"auto","created_at":"2026-02-04 15:09:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2352760,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFluorinated analogues and their pharmacokinetic profiles. a\u003c/strong\u003e, Structural modifications of the fluorinated derivatives. \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eBinding affinities of fluorinated compounds\u003cstrong\u003e \u003c/strong\u003eto native AL deposits. Orange and black arrows indicate the lead compound \u003cstrong\u003e7\u003c/strong\u003e and the fluorinated analogues \u003cstrong\u003eFT-1\u003c/strong\u003e and \u003cstrong\u003eFT-8\u003c/strong\u003e, respectively, both showing nanomolar affinity. \u003cstrong\u003ec\u003c/strong\u003e, Time-activity curves (TACs) of \u003csup\u003e18\u003c/sup\u003eF-labeled tracers in the healthy myocardium of SD rats (male, 6-12 weeks). \u003cstrong\u003ed\u003c/strong\u003e, The SUVRs of blood pool-to-myocardium and lung-to-myocardium at 50-60 min post-injection. \u003cstrong\u003ee\u003c/strong\u003e, MIP images of \u003csup\u003e18\u003c/sup\u003eF-labeled tracers at 60-70 min post-injection. \u003cstrong\u003ef\u003c/strong\u003e, Representative PET images of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8 \u003c/strong\u003ein transverse, sagittal, and coronal planes at 0-1 min, 5-6 min, 10-20 min, and 50-60 min after \u003cem\u003ei.v.\u003c/em\u003e injection. \u003cstrong\u003eg\u003c/strong\u003e, TACs of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8 \u003c/strong\u003ein the lung, liver, and blood pool. Data bars represent the mean ± s.d. (\u003cstrong\u003eb\u003c/strong\u003e, n = 3; \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, n = 2).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7823866/v1/e535fa1c3c455764deb02e81.png"},{"id":101880498,"identity":"2c6daa84-2128-4d23-8cba-9bb84f8791d2","added_by":"auto","created_at":"2026-02-04 15:02:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5476534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAffinity and physicochemical properties of [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]FT-8. a\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eARG images of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8 \u003c/strong\u003ebinding to myocardial slices from patients with AL-CA, ATTR-CA, or healthy controls. Enlarged boxed regions show validation by IF with anti-lambda light chain antibody (ab124719, yellow), anti-transthyretin antibody (11891-1-AP, green), and FS with the cationic tracer \u003cstrong\u003e23\u003c/strong\u003e (red). Scale bars, 2 mm (full) and 100 μm (magnified). \u003cstrong\u003eb\u003c/strong\u003e, ARG images and saturation binding curve of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e on AL-rich myocardial slices (#Case 5-4) at increasing concentrations. ROIs are indicated by red outlines. Data are mean ± s.d. (n = 3). Scale bars, 5 mm. \u003cstrong\u003ec\u003c/strong\u003e, \u003cem\u003eIn vitro\u003c/em\u003e stability of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e in saline and rat plasma. \u003cstrong\u003ed\u003c/strong\u003e, Summary of physicochemical properties and AL binding affinity.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7823866/v1/b6355fe4c104e813cb2becdd.png"},{"id":101787717,"identity":"0c73952d-34ea-4326-89a2-b1f819f76da8","added_by":"auto","created_at":"2026-02-03 15:50:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1945700,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition assays of [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]FT-8 and [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]Florbetazine binding to myocardial slices from CA patients with AL or ATTR pathology.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003eb\u003c/strong\u003e, ARG images of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e and [\u003csup\u003e18\u003c/sup\u003eF]Florbetazine on slices from #Case 5-7 (AL-CA) and #Case 10-3 (ATTR-CA) under increasing ionic strength or in the presence of 500 nM of cold compounds (\u003cstrong\u003eFT-8\u003c/strong\u003e, Florbetapir, PiB, or Florbetazine). \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e, Semiquantitative binding analysis corresponding to panel \u003cstrong\u003ea\u003c/strong\u003e. \u003cstrong\u003ee\u003c/strong\u003e, Semiquantitative binding analysis corresponding to panel \u003cstrong\u003eb\u003c/strong\u003e. ROIs are indicated by red circles. Data are mean ± s.d. (n = 3). Scale bar, 5 mm.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7823866/v1/91807ba03c7b23bf491f0bbe.png"},{"id":101787721,"identity":"89943304-217b-4179-b64f-83f6775d7365","added_by":"auto","created_at":"2026-02-03 15:50:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":6969238,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFirst-in-human PET study of [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]FT-8 in health and disease. a\u003c/strong\u003e, Transverse MIP images of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e in a healthy volunteer at serial time points, with corresponding coronal PET/CT images of the heart. \u003cstrong\u003eb\u003c/strong\u003e, TACs of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8 \u003c/strong\u003ein the myocardium, lung, spleen, kidney, liver, and muscle of the healthy volunteer. \u003cstrong\u003ec\u003c/strong\u003e, MIP images of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8 \u003c/strong\u003ein a patient with AL pathology\u003cstrong\u003e \u003c/strong\u003eacquired 3 hours post-injection, with representative transverse and coronal PET/CT images of the heart. \u003cstrong\u003ed\u003c/strong\u003e, Sagittal PET/CT images of the rectum acquired 3 hours post-injection, alongside ARG of the biopsied specimen and pathological validation using adjacent slices by Congo Red staining under cross-polarized light, FS with permeant cationic tracer \u003cstrong\u003e23\u003c/strong\u003e, and specific IF staining using anti-lambda light chain antibody (ab124719, yellow) and anti-transthyretin antibody (11891-1-AP, green), with nuclear counterstaining by DAPI (blue). Regions of interest are indicated by white arrows in the first panel; region 1 (solid box) and region 2 (dashed box) are shown at higher magnification. Scale bars: 5 mm (full-field) and 100 μm (magnified).\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-7823866/v1/9bde4850ef0cbc799804f95c.png"},{"id":101882961,"identity":"9350db70-ebca-4853-b0bd-78a26cb03632","added_by":"auto","created_at":"2026-02-04 15:26:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":33922059,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7823866/v1/80ad68af-6072-4372-ae40-2d35a19f3995.pdf"},{"id":101787713,"identity":"3ac46e2f-017d-4df5-981f-4567debe011f","added_by":"auto","created_at":"2026-02-03 15:50:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18312,"visible":true,"origin":"","legend":"Clinical trial protocol","description":"","filename":"StudyprotocolforFT8.docx","url":"https://assets-eu.researchsquare.com/files/rs-7823866/v1/e2ccfb1238e2a4c399a2fbef.docx"},{"id":101787723,"identity":"a20877d8-c72b-46b1-a2e0-6ab2ab933bde","added_by":"auto","created_at":"2026-02-03 15:50:28","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1583,"visible":true,"origin":"","legend":"Clinical Trial Summary","description":"","filename":"NCT07130565.csv","url":"https://assets-eu.researchsquare.com/files/rs-7823866/v1/6b3fb76d86858bab7ba2ecf2.csv"},{"id":101787719,"identity":"dd89c928-bc5d-4b6d-afe1-92e88a44a8a5","added_by":"auto","created_at":"2026-02-03 15:50:28","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":27210962,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7823866/v1/24855ebe064b6536c4ef476c.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Protonation-Guided Design and Evaluation of Selective PET Tracers for Light Chain Cardiac Amyloidosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiac amyloidosis (CA) is a progressive infiltrative cardiomyopathy caused by the deposition of amyloid fibrils in the myocardial extracellular matrix, predominantly arising from light chain amyloidosis (AL) or transthyretin amyloidosis (ATTR).\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e AL-CA accounts for over half of all CA cases and stems from misfolded immunoglobulin light chains secreted by abnormal clonal plasma cell proliferation.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Among CA subtypes, AL-CA carries the worst prognosis, with median survival as short as six months, tenfold shorter than that of ATTR-CA.\u003csup\u003e2,4\u003c/sup\u003e Early diagnosis and precise subtyping are essential for guiding treatment, as each subtype follows a distinct clinical course and requires tailored therapies. However, clinical heterogeneity frequently leads to diagnostic delays.\u003c/p\u003e \u003cp\u003eOnce considered rare and usually diagnosed only post-mortem, CA is now increasingly recognized due to heightened awareness and advanced imaging technologies. Autopsy studies have reported CA in up to 43% of individuals aged\u0026thinsp;\u0026ge;\u0026thinsp;75 years, underscoring its underdiagnosis.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Current diagnostic guidelines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) recommend a multimodal approach that integrates electrocardiogram (ECG), echocardiography (Echo), cardiac magnetic resonance (CMR), laboratory testing (e.g., serum/urine analyses and \u003cem\u003eTTR\u003c/em\u003e genotyping), endomyocardial biopsy (EMB), and nuclear imaging with single-photon emission computed tomography (SPECT) or positron emission tomography (PET).\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e While EMB with Congo red staining, immunohistochemistry, and mass spectrometry (MS)-based proteomics remains the diagnostic gold standard, it is invasive, carries procedural risks, and is subject to sampling error (potentially yielding false negatives).\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Non-invasive approaches, although safer, often lack sensitivity, specificity, or the ability to quantify amyloid burden and subtype.\u003c/p\u003e \u003cp\u003eNuclear molecular imaging has transformed the field by enabling non-invasive and longitudinal visualization of disease-associated biomarkers. A major breakthrough was the clinical adoption of bone-avid SPECT tracers such as [\u003csup\u003e99m\u003c/sup\u003eTc]pyrophosphate ([\u003csup\u003e99m\u003c/sup\u003eTc]PYP), [\u003csup\u003e99m\u003c/sup\u003eTc]3,3-diphosphono-1,2-propanodicarboxylic acid ([\u003csup\u003e99m\u003c/sup\u003eTc]DPD), and [\u003csup\u003e99m\u003c/sup\u003eTc]hydroxymethylene diphosphonate ([\u003csup\u003e99m\u003c/sup\u003eTc]HMDP), which are now recommended as non-biopsy diagnostic tools for ATTR-CA.\u003csup\u003e7\u0026ndash;9\u003c/sup\u003e However, [\u003csup\u003e99m\u003c/sup\u003eTc]DPD also demonstrates myocardial uptake in ~\u0026thinsp;40% of AL-CA cases and in rare forms of Apolipoprotein AI amyloidosis, complicating subtype differentiation. \u003csup\u003e10\u0026ndash;12\u003c/sup\u003e PET tracers originally developed for \u003cem\u003eβ\u003c/em\u003e-amyloid (A\u003cem\u003eβ\u003c/em\u003e) imaging in Alzheimer\u0026rsquo;s disease (AD), including [\u003csup\u003e11\u003c/sup\u003eC]PiB, [\u003csup\u003e18\u003c/sup\u003eF]Florbetaben, [\u003csup\u003e18\u003c/sup\u003eF]Florbetapir, [\u003csup\u003e18\u003c/sup\u003eF]Flutemetamol, and [\u003csup\u003e18\u003c/sup\u003eF]Florbetazine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), have also shown affinity for both AL and ATTR fibrils, with higher sensitivity for AL.\u003csup\u003e13\u0026ndash;16\u003c/sup\u003e The only tracer specifically designed for \u003cem\u003epan\u003c/em\u003e-amyloid imaging in CA to date is [\u003csup\u003e124\u003c/sup\u003eI]Evuzamitide, a 45-amino acid peptide carrying 12 net positive charges (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). This agent binds strongly through electrostatic interactions with anionic heparan sulfate proteoglycans (HSPGs), which are colocalized with amyloid deposits and are central to plaque formation.\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Despite its broad amyloid-binding profile, [\u003csup\u003e124\u003c/sup\u003eI]Evuzamitide lacks subtype specificity, necessitating confirmatory laboratory tests for AL-CA. Thus, there remains a pressing need for subtype-specific tracers, particularly for AL-CA, to enable early diagnosis, quantitative assessment, and treatment monitoring.\u003c/p\u003e \u003cp\u003eTo address this challenge, we focused on the distinct molecular features of amyloid fibrils. Compared with TTR, immunoglobulin light chains are more acidic and frequently N-glycosylated, generating localized negative charges along the \u003cem\u003eβ\u003c/em\u003e-strand axis.\u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e These unique features provide opportunities for selective targeting with small-molecule cations. We hypothesize that positively charged molecules could first localize to acidic residues and N-glycosylation sites through electrostatic attraction, and subsequently engage hydrophobic \u003cem\u003eβ\u003c/em\u003e-sheet cavities via combined electrostatic, hydrophobic, hydrogen-bonding, and π-π stacking interactions. This mechanism differs from [\u003csup\u003e124\u003c/sup\u003eI]Evuzamitide, which primarily targets HSPGs. Supporting this model, our previously reported tracer [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003e23\u003c/b\u003e selectively bound AL deposits (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), validating the complementary roles of electrostatic surface interactions and conformational \u003cem\u003eβ\u003c/em\u003e-sheet cavity recognition.\u003c/p\u003e \u003cp\u003eA major limitation of permanently cationic compounds is their high baseline cardiac uptake, which results from pronounced mitochondrial trapping in cardiomyocytes. For instance, [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003e23\u003c/b\u003e (log D\u003csub\u003e7.4\u003c/sub\u003e = 0.22) showed undesired retention in healthy myocardium.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Enhancing hydrophilicity or employing a dynamic protonation strategy could reduce off-target accumulation while preserving affinity.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTo this end, we selected piperazine, a privileged N-heterocyclic scaffold widely used in drug development, for its synthetic versatility and tunable protonation states.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Pyridyl substituents incorporated into the diarylpiperazine scaffold exhibit position-dependent p\u003cem\u003eK\u003c/em\u003ea variation, allowing fine-tuning of molecular charge to meet the desired physicochemical and biological properties. The piperazine ring also provides dual hydrogen-bonding with AL fibrils, while appended aromatic rings further contribute hydrophobic and π-π stacking interactions with \u003cem\u003eβ\u003c/em\u003e-sheets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eTo test this dynamic protonation design, we synthesized 4-pyridyl, 2-pyridyl, and phenyl analogues representing high-, low-, and non-protonated forms. Their selectivity was systematically evaluated using autoradiography (ARG) on human myocardial tissues from autopsies and biopsies. The highly protonated 4-pyridylpiperazine scaffold was strategically diversified into nine fluorinated derivatives, which were comprehensively characterized through \u003cem\u003ein vitro\u003c/em\u003e binding assays, mechanistic investigations, \u003cem\u003ein vivo\u003c/em\u003e PET pharmacokinetics in rodents, and first-in-human PET imaging studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and discussion","content":"\u003ch2\u003eN-Protonation governs selective recognition of AL deposits\u003c/h2\u003e\n\u003cp\u003eGiven the advantageous properties of \u003csup\u003e125\u003c/sup\u003eI-labeled probes, including their long half-life (T\u003csub\u003e1/2\u003c/sub\u003e = 59.4 d) and high achievable molar activity (\u003cem\u003eA\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e = 81.4 GBq/\u0026mu;mol), which allow reliable detection of probe-target interactions in pathological specimens at extremely low concentrations by \u003cem\u003ein vitro\u003c/em\u003e ARG. Guided by this rationale, we initially designed and synthesized three iodinated compounds (\u003cstrong\u003e7\u003c/strong\u003e-\u003cstrong\u003e9\u003c/strong\u003e) with distinct N-protonation states under physiological conditions (pH = 7.4). As outlined in Supplementary Scheme 1, the diarylpiperazine derivatives were effectively constructed in a one-step reaction via either Buchwald-Hartwig cross-coupling or ethylene glycol-mediated arylamination between amine and halides. The synthetic route involved an initial preparation of brominated intermediates, followed by Stille cross-coupling and subsequent iodination to produce the cold compounds \u003cstrong\u003e7\u003c/strong\u003e-\u003cstrong\u003e9\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewith overall yields of 8 - 29% and purities exceeding 95% (Supplementary Fig. 1 and Supplementary Table 2).\u003c/p\u003e\n\u003cp\u003eMeasured p\u003cem\u003eK\u003c/em\u003ea values revealed that the nitrogen atoms directly attached to the iodine-substituted aromatic rings remained unprotonated at physiological pH (7.4), as reflected by uniformly low p\u003cem\u003eK\u003c/em\u003ea\u003csub\u003e1\u003c/sub\u003e values of 3.69, 3.54, and 3.98 (Fig. 2a). Instead, protonation occurred predominantly on the nitrogen atom located on the opposite aromatic ring. Among these compounds, the 4-pyridyl analogue \u003cstrong\u003e7\u003c/strong\u003e displayed the highest basicity (p\u003cem\u003eK\u003c/em\u003ea\u003csub\u003e2\u003c/sub\u003e = 8.97), resulting in near-complete protonation (97.40%). In contrast, the 2-pyridyl analogue \u003cstrong\u003e8\u003c/strong\u003e showed only marginal protonation (2.03%), whereas the phenyl analogue \u003cstrong\u003e9\u003c/strong\u003e remained entirely unprotonated. Thus, these three compounds served\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eas representative prototypes of high-, low-, and non-protonated small molecules, distinguished by the position or absence of a nitrogen atom.\u003c/p\u003e\n\u003cp\u003eRadiolabeling of [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7-9\u003c/strong\u003e was achieved by iododestannylation with radiochemical yields (RCYs) of 66-83% and radiochemical purities (RCPs) over 95%, as verified by co-injection with corresponding cold compounds (Supplementary Fig. 2 and Supplementary Table 3). ARG on postmortem human myocardial slices was then conducted to evaluate their binding characteristics. [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e demonstrated strong and selective binding in AL-positive samples, with only background signals from ATTR-positive and control tissues. By contrast, [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e9\u003c/strong\u003e failed to differentiate between AL and ATTR deposits, resembling the behavior of established A\u003cem\u003e\u0026beta;\u003c/em\u003e-PET tracers (Fig. 2a). [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e8\u003c/strong\u003e exhibited similar labeling of both AL and ATTR pathologies, consistent with its low\u003cem\u003e\u0026nbsp;\u003c/em\u003edegree of N-protonation. Semi-quantitative analysis of the autoradiographic images further revealed a protonation-dependent selectivity, with tracer preference for AL over ATTR deposits positively correlating with the extent of protonation across the diarylpiperazine series (Fig. 2b). Notably, [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e displayed a 5-fold selectivity for AL relative to ATTR, whereas [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e8\u003c/strong\u003e yielded a near-equivalent selectivity ratio (1.20), and the non-protonatable [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e9\u003c/strong\u003e exhibited the lowest ratio of 0.65. These findings provide initial validation for a protonation-guided tracer design strategy for selective targeting of cardiac AL deposits.\u003c/p\u003e\n\u003ch2\u003eBinding characteristics of hyper-protonated forms\u003c/h2\u003e\n\u003cp\u003eTo elucidate the mechanism underlying the selective recognition of myocardial amyloid by these diarylpiperazine tracers, we next systematically characterized probe-target interactions. Given that protonated microspecies may preferentially associate with AL fibrils through electrostatic attraction, competitive binding assays were performed under increased ionic strength to disturb electrostatic contributions. In AL-rich regions, [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e binding was markedly reduced, with a 94.67% \u0026plusmn; 0.18% decrease in the presence of 1.0 M NaCl (Figs. 2c-2d and Supplementary Fig. 4a), whereas no ionic strength-dependent effects were observed for [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e8\u003c/strong\u003e or [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e9\u003c/strong\u003e. Likewise, the binding of [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e8\u003c/strong\u003e to ATTR deposits remained unaffected by ionic strength variations (Figs. 2g-2h and Supplementary Figs. 7-8), indicating that electrostatic interaction is the dominant determinant of selectively targeting AL deposits.\u003c/p\u003e\n\u003cp\u003eHydrophobic interactions represent another major driving force in amyloid recognition. An 8-hour formic acid pretreatment, known to disrupt \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet structures, abolished the specific [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e signals on AL-rich myocardial slices (Figs. 2e-2f). Compound \u003cstrong\u003e7\u003c/strong\u003e at 500 nM achieved 65.65% \u0026plusmn; 1.10% self-inhibition, and as anticipated, formic acid treatment reduced [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u0026nbsp;\u003c/strong\u003ebinding to background levels (77.34% \u0026plusmn; 2.80% reduction). This result provides direct evidence that hydrophobic interactions with \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet cavities are indispensable for AL recognition. On AL-enriched slices, co-incubation with 500 nM of unlabeled compounds \u003cstrong\u003e7\u003c/strong\u003e-\u003cstrong\u003e9\u003c/strong\u003e effectively inhibited [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e8\u003c/strong\u003e binding by 78.50% \u0026plusmn; 2.66%, 90.97% \u0026plusmn; 0.28%, and 86.91% \u0026plusmn; 0.13%, respectively (Fig. 2i and Supplementary Fig. 7). A similar inhibition profile was also observed for [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e9\u0026nbsp;\u003c/strong\u003e(Supplementary Fig. 8), indicating co-occupancy of compounds \u003cstrong\u003e7\u003c/strong\u003e-\u003cstrong\u003e9\u003c/strong\u003e within the \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet cavity of AL fibrils. At the same concentration, established A\u003cem\u003e\u0026beta;\u003c/em\u003e-PET tracers (Florbetazine, PiB, and Florbetapir) also inhibited [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e8\u003c/strong\u003e and [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e9\u003c/strong\u003e binding with an average efficiency of 70.31% \u0026plusmn; 6.80% and 74.23% \u0026plusmn; 4.87%. Notably, the diarylpiperazine compounds \u003cstrong\u003e7\u003c/strong\u003e-\u003cstrong\u003e9\u003c/strong\u003e exhibited significantly stronger inhibition than these planar, conjugated A\u003cem\u003e\u0026beta;\u0026nbsp;\u003c/em\u003etracers, suggesting that the piperazine linker may enhance binding within the AL fibril pocket.\u003c/p\u003e\n\u003cp\u003eIn contrast, competition assays on ATTR-rich slices revealed markedly different inhibition patterns. At 500 nM, compound \u003cstrong\u003e7\u003c/strong\u003e failed to displace either [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e8\u003c/strong\u003e or [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e9\u003c/strong\u003e from ATTR deposits (Fig. 2j and Supplementary Fig. 8). In comparison, compounds \u003cstrong\u003e8\u003c/strong\u003e and \u003cstrong\u003e9\u003c/strong\u003e at the same concentration reduced tracer binding by more than 50%. These findings, consistent with ARG results, suggest that while the diarylpiperazine scaffold adopts a conformation compatible with ATTR recognition, the high-protonated 4-pyridyl substituent in compound \u003cstrong\u003e7\u003c/strong\u003e may hinder such interactions owing to repulsion from the positively charged basic residues of TTR. Furthermore, 500 nM of Florbetazine inhibited ATTR binding of [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e8\u003c/strong\u003e and [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e9\u003c/strong\u003e by 58.77% \u0026plusmn; 1.18% and 34.82% \u0026plusmn; 0.77%, respectively, whereas PiB and Florbetapir produced minimal inhibition, in line with previous reports.\u003csup\u003e16,31\u003c/sup\u003e These results further reinforce the notion that the diarylpiperazine scaffold provides substantial potential for hydrophobic interactions within \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet cavities.\u003c/p\u003e\n\u003cp\u003eTogether, these experimental results provide preliminary support for our hypothesis that AL aggregates are more negatively charged along the \u003cem\u003e\u0026beta;\u003c/em\u003e-strand axis than ATTR aggregates, likely reflecting a higher prevalence of acidic residues and N-glycosylation modifications. Such electrostatic landscapes promote the enrichment of \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet-recognizing molecules bearing positive charges, whether permanently cationic or protonatable, around AL deposits, followed by hydrophobic engagement with AL fibrils. Of particular note, the binding mechanism of the N-protonated tracer [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e differs from that of the cationic polypeptide tracer [\u003csup\u003e124\u003c/sup\u003eI]Evuzamitide described in the introduction, even though the latter also displayed pronounced inhibition (~ 90%) under high ionic strength (1.0 M NaCl).\u003csup\u003e32\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eTo further validate the favorable properties of [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u0026nbsp;\u003c/strong\u003eacross the heterogeneity of light-chain sequences in pathological settings, additional myocardial specimens enriched with pathological confirmed AL or ATTR deposits by anti-lambda light chain antibody or anti-transthyretin antibody were examined by \u003cem\u003ein vitro\u003c/em\u003e ARG (specimen details in Supplementary Table 1).\u003csup\u003e26,33\u003c/sup\u003e [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e exhibited diffuse or focally increased uptake across multiple AL cases, including three autopsy and three biopsy specimens. Regions labeled by [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e showed excellent co-localization with fluorescence staining (FS) using the reference tracer \u003cstrong\u003e23\u003c/strong\u003e (Fig. 3). By contrast, [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e produced only uniform, weak signals in ATTR-rich slices, a pattern distinct from the profiles revealed by anti-transthyretin IF (Fig. 4). These findings consistently demonstrated the high selectivity and sensitivity of [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e for AL over ATTR and non-amyloid controls.\u003c/p\u003e\n\u003cp\u003eQuantitative self-displacement studies were then performed on adjacent myocardial slices using [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u0026nbsp;\u003c/strong\u003eas the radioligand. Analysis of radioactivity density (Digital Light Units/mm\u0026sup2;, DLU/mm\u0026sup2;) within AL-rich regions generated a displacement curve that yielded an IC\u003csub\u003e50\u003c/sub\u003e value of 10.62 \u0026plusmn; 3.22 nM, indicating high affinity for AL deposits (Supplementary Fig. 5).\u003c/p\u003e\n\u003ch2\u003eConversion into \u003csup\u003e18\u003c/sup\u003eF-labeled AL-PET tracers\u003c/h2\u003e\n\u003cp\u003eTo extend the application of PET imaging for the selective diagnosis of AL-CA, we adopted a dual structural modification strategy to generate fluorinated 4-pyridylpiperazine derivatives as candidate AL-targeted tracers (Fig. 5a). Direct replacement of the iodine atom in lead compound \u003cstrong\u003e7\u003c/strong\u003e with fluorine yielded analogue\u003cstrong\u003e\u0026nbsp;FT-1\u003c/strong\u003e, which retained high affinity for native AL deposits in competitive binding assays with [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e7\u003c/strong\u003e (IC\u003csub\u003e50\u003c/sub\u003e = 12.87 \u0026plusmn; 2.54 nM; Fig. 5b and Supplementary Fig. 6). However, attempts to radiolabel \u003cstrong\u003eFT-1\u0026nbsp;\u003c/strong\u003evia its aryl-BPin precursor using Cu(py)\u003csub\u003e4\u003c/sub\u003e(OTf)\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ecatalysis in TBAB/DMA/n-BuOH at 110 \u0026deg;C gave a RCY of only 0.6%, precluding\u0026nbsp;further evaluation. A benzene analogue (\u003cstrong\u003eFT-2\u003c/strong\u003e) also required aryl-BPin precursor, rendering it similarly suboptimal for large-scale preparation.\u003c/p\u003e\n\u003cp\u003eWe therefore pursued an alternative \u003csup\u003e18\u003c/sup\u003eF-labeling approach using trimethylammonium trifluoroacetate salts as precursors, which are more compatible with mild conditions. Through Buchwald-Hartwig arylamination, we synthesized \u003cstrong\u003eFT-3\u003c/strong\u003e and \u003cstrong\u003eFT-4\u003c/strong\u003e in 22% and 43% yield, respectively. Both compounds showed moderately reduced affinities (IC\u003csub\u003e50\u003c/sub\u003e = 64.27 \u0026plusmn; 21.55 nM and 240.40 \u0026plusmn; 67.01 nM, respectively). Notably, the \u003cem\u003epara\u003c/em\u003e-fluorinated analogue \u003cstrong\u003eFT-1\u003c/strong\u003e exhibited higher affinity than its \u003cem\u003emeta\u003c/em\u003e-fluorinated counterpart \u003cstrong\u003eFT-4\u003c/strong\u003e, suggesting a preference for the \u003cem\u003epara\u003c/em\u003e-substitution site in AL binding. Despite the moderate affinities, \u003cem\u003ein vitro\u003c/em\u003e ARG confirmed the selective binding of both tracers to AL deposits (Supplementary Fig. 11a). Importantly, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-3\u003c/strong\u003e and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-4\u003c/strong\u003e were radiolabeled with substantially improved RCYs (9%-11%, non-decay-corrected) using Kryptofix\u003csub\u003e222\u003c/sub\u003e/K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e in anhydrous acetonitrile at 80 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003eWe next introduced \u003csup\u003e18\u003c/sup\u003eF at terminal alkyl positions via nucleophilic aliphatic substitution, an efficient route for radiolabeling. Through tailored synthetic methods, several side chains, including 2-fluoroethanol, chiral 3-fluoropropan-2-ol, and chiral 4-fluorobutane-2,3-diol, were incorporated into the aromatic system to yield analogues \u003cstrong\u003eFT-5\u003c/strong\u003e to \u003cstrong\u003eFT-9\u0026nbsp;\u003c/strong\u003e(Supplementary Scheme 2).\u003csup\u003e34\u003c/sup\u003e In line with structure-activity relationship (SAR) trends for A\u003cem\u003e\u0026beta;\u003c/em\u003e tracers, 2-fluoroethoxy substitution preserved favorable AL binding, whereas additional hydroxyl groups in (\u003cem\u003eS\u003c/em\u003e)-3-fluoro-2-hydroxypropanoxy and ((2\u003cem\u003eR\u003c/em\u003e,3\u003cem\u003eS\u003c/em\u003e)-4-fluoro-2,3-dihydroxybutanoxy substituents weakened this interaction (Fig. 5b).\u003csup\u003e35\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAs anticipated, the \u003cem\u003emeta\u003c/em\u003e-substituted 2-fluoroethoxy analogue \u003cstrong\u003eFT-9\u003c/strong\u003e exhibited the lowest affinity (IC\u003csub\u003e50\u003c/sub\u003e \u0026gt; 1000 nM), whereas the \u003cem\u003epara\u003c/em\u003e-substituted \u003cstrong\u003eFT-5\u003c/strong\u003e and \u003cstrong\u003eFT-8\u003c/strong\u003e displayed acceptable potency (IC\u003csub\u003e50\u003c/sub\u003e = 68.38 \u0026plusmn; 14.95 nM and 11.61 \u0026plusmn; 1.32 nM, respectively). Their tosylated precursors were efficiently radiofluorinated with RCYs up to 33% (non-decay corrected, Supplementary Scheme 3). All \u003csup\u003e18\u003c/sup\u003eF-labeled tracers showed RCP \u0026gt;95% after HPLC purification and were verified by co-injection with corresponding \u003csup\u003e19\u003c/sup\u003eF standards (Supplementary Fig. 3 and Supplementary Table 3), supporting their suitability for further bio-evaluation.\u003c/p\u003e\n\u003ch2\u003eMicro-PET pharmacokinetics in rats\u003c/h2\u003e\n\u003cp\u003eNext, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-3\u003c/strong\u003e-[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-5\u003c/strong\u003e, and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e were prioritized for \u003cem\u003ein vivo\u003c/em\u003e pharmacokinetic evaluation in rodents. Following intravenous injection (\u003cem\u003ei.v.\u003c/em\u003e), all\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003etracers\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003erapidly entered systemic circulation, with immediate cardiac passage and the emergence of clear myocardial contours within one minute post-injection. The mean standard uptake value (SUV\u003csub\u003emean\u003c/sub\u003e) reached a peak of 5.03, 5.91, 4.94, and 5.60 for [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-3\u003c/strong\u003e-[\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-5\u003c/strong\u003e and [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e, respectively (Fig. 5c). Pulmonary uptake was observed in parallel, with [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-4\u003c/strong\u003e showing the highest transient accumulation (SUV\u003csub\u003epeak\u003c/sub\u003e = 7.41) before redistribution (Fig. 5g and Supplementary Figs. 9d-9f).\u003c/p\u003e\n\u003cp\u003eSubsequently, all tracers underwent rapid myocardial and pulmonary clearance, approaching background levels at 50-60 min, indicative of negligible non-specific retention in healthy tissues. Notably, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u0026nbsp;\u003c/strong\u003edisplayed a delayed myocardial clearance during the first 10 minutes, but the SUV ratio (SUVR) between myocardium and anatomically adjacent tissues, particularly lung and blood pool, approached close 1.0 at 50-60 min, satisfying the requirements for cardiac imaging (Fig. 5d).\u003c/p\u003e\n\u003cp\u003eDynamic PET and maximum intensity projection (MIP, 60-70 min post-injection) analyses of these tracers revealed predominant hepatobiliary clearance (Figs. 5e-5f and Supplementary Figs. 9a-9c), in line with other lipophilic tracers. Only [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-4\u0026nbsp;\u003c/strong\u003eshowed transient hepatic accumulation (SUV\u003csub\u003emean\u003c/sub\u003e = 6.15 at 14-15 min post-injection), followed by partial clearance (SUV\u003csub\u003emean\u003c/sub\u003e = 4.72 at 50-60 min), slightly below [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u0026nbsp;\u003c/strong\u003e(SUV\u003csub\u003emean\u003c/sub\u003e = 4.87 at 50-60 min, Supplementary Fig. 10). However, mild defluorination signals were observed in joints and thoracic\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003evertebrae for [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eOverall, all radiolabeled candidates demonstrated favorable myocardial pharmacokinetics, but [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e emerged as the most suitable lead, characterized by rapid myocardial uptake (SUV\u003csub\u003epeak\u003c/sub\u003e = 5.60), efficient washout (washout\u003csub\u003e1 min/60 min\u003c/sub\u003e = 8.26), minimal residual activity in the blood-pool (SUV\u003csub\u003emean\u003c/sub\u003e = 0.53 at 50-60 min) and lungs (SUV\u003csub\u003emean\u003c/sub\u003e = 0.85 at 50-60 min), and the strongest AL-binding potency. Physiological hepatic and renal uptake did not interfere with cardiac visualization, consistent with previously reported small-molecule CA tracers.\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eFor subsequent clinical translation, large-scale automated radiosynthesis is indispensable. As summarized in Fig. 6d, automated synthesis of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e achieved a non-decay corrected conversion yield of 30% and a molar activity of 159.62 GBq/\u0026mu;mol. Additionally, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e exhibited excellent stability in both saline and rat plasma, maintaining integrity (\u0026gt; 98%) for at least 8 hours in saline and 5 hours in rat plasma at 37 \u0026deg;C (Fig. 6c). No bone uptake was detected in living animals, further confirming its\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e stability. These profiles collectively support the feasibility of advancing [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u0026nbsp;\u003c/strong\u003einto clinical studies. UV-metric titration determined the p\u003cem\u003eK\u003c/em\u003ea values of the aromatic nitrogen atoms in [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e to be 2.97 and 8.76, corresponding to a dynamic protonation fraction of 95.85% under physiological conditions. Although the measured log D\u003csub\u003e7.4\u0026nbsp;\u003c/sub\u003ewas 1.11 \u0026plusmn; 0.10, \u003cem\u003ein vivo\u003c/em\u003e studies in rodents indicated that the equilibrium between protonated and deprotonated states minimizes undesirable electrostatic interactions with negatively charged intracellular components. This balance enhances membrane permeability and enables [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e to selectively engage AL deposits in myocardial tissue without binding to mitochondria in healthy myocardium.\u003c/p\u003e\n\u003ch2\u003eBinding of [\u003csup\u003e18\u003c/sup\u003eF]FT-8 to human AL pathologies\u003c/h2\u003e\n\u003cp\u003eThe binding selectivity of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u0026nbsp;\u003c/strong\u003ewas validated by \u003cem\u003ein vitro\u003c/em\u003e ARG studies on an expanded cohort of myocardial samples, including six from patients with AL-CA, six with ATTR-CA, and two healthy controls. As shown in Fig. 6a, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e produced high-intensity signals across all AL samples, with distribution patterns consistent with staining by an anti-lambda light-chain antibody and\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethe cationic tracer \u003cstrong\u003e23\u003c/strong\u003e. In contrast, only background-level signals were detected on slices from ATTR and healthy hearts. Saturation binding assays further confirmed the high affinity of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u0026nbsp;\u003c/strong\u003efor AL deposits, yielding a \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e of 114.81 nM in case 5-6 (Fig. 6b). Moreover, a bone marrow specimen from a patient diagnosed with plasmacytoma and light chain restriction, a condition pathologically distinct but closely related to AL, also showed strong and specific [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u0026nbsp;\u003c/strong\u003ebinding\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Supplementary Fig. 11b). These findings highlight the potential diagnostic utility of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u0026nbsp;\u003c/strong\u003enot only in cardiac amyloidosis but also in extracardiac light chain associated pathologies.\u003c/p\u003e\n\u003cp\u003eIn the presence of 500 nM \u003cstrong\u003eFT-8\u003c/strong\u003e, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e binding was reduced by 80.56% \u0026plusmn; 1.62% in AL-enriched regions, whereas only background-level signals persisted in areas of extensive transthyretin deposits (Figs. 7a, 7c, and Supplementary Fig. 4b). These self-block assays confirm that [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u0026nbsp;\u003c/strong\u003ebinds selectively and specifically to AL deposits.\u003c/p\u003e\n\u003cp\u003eWhen challenged with increasing ionic strength, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e exhibited marked sensitivity, with AL bound radioactivity declining sharply to 21.88% \u0026plusmn; 3.17% at 0.1 M NaCl and stabilizing at higher concentrations (Fig. 7d). By contrast, [\u003csup\u003e18\u003c/sup\u003eF]Florbetazine binding was unaffected by NaCl concentrations up to 5 M, similar to [\u003csup\u003e125\u003c/sup\u003eI]\u003cstrong\u003e8\u003c/strong\u003e-\u003cstrong\u003e9\u003c/strong\u003e, and the elevated signals observed in myocardial slices were attributable to salt precipitation (Fig. 7b). This differential behavior underscores the dominant contribution of the protonated microspecies (95.85%) of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e, which confers strong electrostatic avidity for AL binding.\u003c/p\u003e\n\u003cp\u003eConsistent with expected hydrophobic interactions with \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet structures, A\u003cem\u003e\u0026beta;\u003c/em\u003e-PET agents (PiB, Florbetapir, and Florbetaben) produced an average of 24.55% inhibition of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e to AL binding. Notably, when [\u003csup\u003e18\u003c/sup\u003eF]Florbetazine was used as the radioligand, unlabeled \u003cstrong\u003eFT-8\u003c/strong\u003e suppressed AL binding by 59.24% \u0026plusmn; 1.60%, within the same range as inhibition by cold Florbetazine (48.13% \u0026plusmn; 4.89%), while showing no blocking effect against ATTR deposits (Fig. 7e). These results suggest that \u003cstrong\u003eFT-8\u003c/strong\u003e engages strong hydrophobic interactions on par with reported tracers, but such contacts represent secondary, weaker forces in ATTR deposits. The dominant mechanism of AL recognition by [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u0026nbsp;\u003c/strong\u003eremains electrostatic avidity, which cannot be inhibited by unprotonated A\u003cem\u003e\u0026beta;\u003c/em\u003e-PET tracers.\u003c/p\u003e\n\u003ch2\u003eFirst-in-human PET study of\u0026nbsp;[\u003csup\u003e18\u003c/sup\u003eF]FT-8\u003c/h2\u003e\n\u003cp\u003eEncouraged by the preclinical data demonstrating the high affinity, selectivity, and favorable kinetics of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e in rodents, we performed an exploratory first-in-human PET study to characterize its pharmacokinetics and evaluate its potential to visualize AL deposits in a patient with systemic amyloidosis. [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u0026nbsp;\u003c/strong\u003ewas well tolerated, with no adverse events reported following tracer administration in either subject.\u003c/p\u003e\n\u003cp\u003eIn the healthy volunteer, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e rapidly accumulated in the myocardium, peaking at an SUV\u003csub\u003emean\u003c/sub\u003e of 7.04 within 10 minutes post-injection (Figs. 8a-8b). Uptake was also observed in the intestine, kidneys, spleen, liver, lungs, brain, salivary glands, thyroid, and parotid gland. With the exception of the liver and intestine, tracer activity in all organs declined over time, parallel to blood clearance, generally consistent with rodent kinetics. As shown in Fig. 8b, myocardial uptake decreased by 45% within the first hour, reaching an SUV\u003csub\u003emean\u003c/sub\u003e of 2.40 at 3 h post-injection (clearance ratio 2.94). Spleen and lung activity similarly diminished, yielding weak signals after 3 hours (SUV\u003csub\u003emean\u003c/sub\u003e = 1.78 and 1.00, respectively). By contrast, persistent liver and intestinal uptake precluded imaging of these regions. Notably, the skeletal system showed only background activity, with no evidence of \u003cem\u003ein vivo\u003c/em\u003e defluorination. Collectively, these results identify 3 h post-injection as an optimal imaging window, offering a favorable background for target visualization.\u003c/p\u003e\n\u003cp\u003eOn the basis of this biodistribution profile in healthy volunteer, we assessed organ-specific AL binding in a patient with systemic amyloidosis using PET/CT acquired at 3 h post-injection (Fig. 8c). [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e showed pronounced and heterogeneous uptake in AL-burdened organs, including the left and right ventricular walls (SUV\u003csub\u003emean\u003c/sub\u003e = 7.44), lungs (SUV\u003csub\u003emean\u003c/sub\u003e = 6.77), spleen (SUV\u003csub\u003emean\u003c/sub\u003e = 6.63), rectum (SUV\u003csub\u003emean\u003c/sub\u003e = 4.63), and tongue (SUV\u003csub\u003emean\u003c/sub\u003e = 2.82), clearly distinguishing the patient from the healthy control. A rectal biopsy specimen confirmed amyloid deposits by Congo Red staining under cross-polarized light and FS using the cationic tracer \u003cstrong\u003e23\u0026nbsp;\u003c/strong\u003e(Fig. 8d). Specifically, IF analysis further identified the deposits as lambda light chain-derived, with positive anti-lambda and negative anti-transthyretin antibody signals. The concordance between \u003cem\u003ein vivo\u003c/em\u003e uptake and \u003cem\u003eex vivo\u003c/em\u003e pathology supports [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e as a specific AL tracer.\u003c/p\u003e\n\u003cp\u003eAlthough bone-avid tracers have utility in ATTR amyloidosis, their mechanism of selectivity, binding to microcalcifications, can lead to false positives in AL cases.\u003csup\u003e36\u003c/sup\u003e Indeed, about 40% of AL patients in a cohort of 292 exhibited [\u003csup\u003e99m\u003c/sup\u003eTc]DPD uptake in the myocardium, underscoring the risk of misclassification.\u003csup\u003e11\u003c/sup\u003e Current diagnostic pathways for ATTR require prior exclusion of AL, yet no selective radiotracers exist for AL itself, necessitating invasive biopsy. The ability of [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e to identify AL deposits non-invasively thus represents a major advance, with potential to enable accurate differentiation of CA subtypes and to reduce reliance on tissue sampling.\u003c/p\u003e\n\u003cp\u003eCollectively, these first-in-human results demonstrate that [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e imaging can provide a safe, non-invasive means to detect both cardiac and extracardiac AL involvement, warranting further validation in larger cohorts and in ATTR populations.\u003c/p\u003e\n\u003ch2\u003eOutlook\u003c/h2\u003e\n\u003cp\u003eGiven the aggressive progression of AL-CA, precise subtyping following early diagnosis is crucial for achieving the best outcomes. Future clinical practice is likely to demand diagnostic approaches that are simpler, more accurate, and non-invasive, with selective PET tracers emerging as powerful enablers.\u003c/p\u003e\n\u003cp\u003eDistinct from conventional screening pipelines based on synthetic fibrils or animal models, the use of native amyloid deposits from patient myocardium is expected to preserve authentic conformations and post-translational modifications. This approach provides a clinically relevant substrate that can accelerate probe discovery, improve reliability, and validate translational potential.\u003c/p\u003e\n\u003cp\u003eProtonation-driven recognition is emerging as a rational design principle with broad translational potential. At the tissue-level, hyper-protonated microspecies selectively bind AL deposits through electrostatic interactions with negatively charged amino acid residues and glycosylated motifs on fibril surfaces, reinforced by hydrophobic and hydrogen-bonding contacts with \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet structures. The optimized tracer, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eexhibits predominant affinity, selectivity, and favorable kinetics, and can be readily synthesized in hospital settings. A single PET scan at 3 h post-injection achieves clear visualization of AL deposits without evidence of \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003edefluorination. Compared with [\u003csup\u003e124\u003c/sup\u003eI]Evuzamitide, which suffers from long half-life, deiodination with free iodine accumulation in the thyroid (requiring potassium iodide prophylaxis), and delayed imaging kinetics, [\u003csup\u003e18\u003c/sup\u003eF]\u003cstrong\u003eFT-8\u003c/strong\u003e offers lower radiation exposure, simplified workflows, and a cleaner thyroidal background, enabling detection of rare thyroid involvement. Because AL amyloidosis frequently involves multiple extracardiac organs, this new tracer further enables simultaneous assessment of cardiac and systemic disease within a single scan. Its safety, dosimetry, sensitivity, and specificity are now being evaluated in larger clinical cohorts.\u003c/p\u003e\n\u003cp\u003eChallenges remain in interpreting signals from the liver and intestine, which reflect high uptake and warrant further tracer optimization. Nonetheless, the framework established here provides a foundation for the development of new-generation tracers with preserved affinity and improved pharmacokinetics, and mature radiotracers could ultimately reshape the diagnostic pathway for CA. More broadly, the protonation-guided design strategy may extend beyond AL. ATTR protein, which is less acidic, may be more amenable to neutral or anionic ligands, whereas hyperphosphorylated tau protein in the brain may preferentially engage protonated tracers. Thus, protonation-sensitive probe design offers a generalizable framework for targeting proteins with defined charge-distribution patterns across a spectrum of diseases.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChemistry and radiochemistry\u003c/h2\u003e \u003cp\u003eSynthetic procedures, purity analyses, and characterization data are provided in the Supplementary Information.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAll procedures involving animals were approved by the Animal Care Committee of Beijing Normal University (Approval No. BNUCC-EAW-2023-0615-01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHuman participants\u003c/h2\u003e \u003cp\u003eThe first-in-human PET/CT study was approved by the Ethics Committee of Tianjin Medical University General Hospital (Approval No. IRB2023-YX-239-01) and registered at \u003cem\u003eClinicalTrials.gov\u003c/em\u003e (NCT07232459). Written informed consent was obtained from the participants or their caregivers. This study included one healthy volunteer (41 years old, 82 kg, male) and one patient diagnosed with systemic amyloidosis (71 years old, 53 kg, male).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eARG\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFormalin-fixed paraffin-embedded myocardial slices were deparaffinized in xylene (10 min) and rinsed in EtOH. Pre-treated slices were incubated with either \u003csup\u003e125\u003c/sup\u003eI-labeled tracers (74 kMq/mL, 2 h) or \u003csup\u003e18\u003c/sup\u003eF-labeled tracers (0.74 MBq/mL, 1 h) at room temperature. Unbound tracer was removed by a rapid ethanol rinse and 50% ethanol washing (5 min). Slices were dried and exposed to a phosphor screen (10 h for \u003csup\u003e125\u003c/sup\u003eI-labeled tracers; 1 h for \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-labeled tracers). ARG images were acquired on a Cyclone Plus storage phosphor system (PerkinElmer, USA) and quantified using OptiQuant (PerkinElmer, USA) and ImageJ.\u003c/p\u003e \u003cp\u003eSelectivity for AL versus ATTR aggregates was calculated as the following formula,\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{S}\\text{e}\\text{l}\\text{e}\\text{c}\\text{t}\\text{i}\\text{v}\\text{i}\\text{t}\\text{y}\\:=\\frac{{AL}^{+}}{{ATTR}^{+}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{AL}^{+}\\)\u003c/span\u003e\u003c/span\u003e/\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{ATTR}^{+}\\)\u003c/span\u003e\u003c/span\u003e was the main grey value with AL/ATTR pathology in red/blue circles (indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) calculated by ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBinding potency to AL deposits\u003c/h2\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e values were measured from ARG studies of serial myocardial slices from an AL-CA case. For IC\u003csub\u003e50\u003c/sub\u003e, pre-treated slices were incubated with a mixture of [\u003csup\u003e125\u003c/sup\u003eI]\u003cb\u003e7\u003c/b\u003e (74 kBq/mL) and increasing concentrations of unlabeled competitor (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e \u0026minus;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003e M) for 2 h at room temperature. For \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e, pre-treated slices were incubated with a solution of gradient concentration of [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eFT-8\u003c/b\u003e (0.23, 2.32, 11.6, 23.2, 58.0, 115.9, 231.8, 463.6 nM) for 1 h at room temperature. The washing, ARG image acquisition, and analysis were performed as described above in the \u003cem\u003ein vitro\u003c/em\u003e ARG.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInhibition assays\u003c/h2\u003e \u003cp\u003eThe inhibition ratio was measured from ARG studies of serial myocardial slices. Pretreated slices were incubated with solutions of [\u003csup\u003e125\u003c/sup\u003eI]\u003cb\u003e7\u003c/b\u003e\u0026ndash;\u003cb\u003e9\u003c/b\u003e (74 kBq/mL), [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eFT-8\u003c/b\u003e (0.74 MBq/mL), or [\u003csup\u003e18\u003c/sup\u003eF]Florbetazine (0.56 MBq/mL) in the presence of different concentrations of competing compounds (2 h for \u003csup\u003e125\u003c/sup\u003eI-labeled tracers; 1 h for \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-labeled tracers) at room temperature. The washing, ARG image acquisition, and analysis were performed as described above in the \u003cem\u003ein vitro\u003c/em\u003e ARG.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDistribution coefficient determination\u003c/h2\u003e \u003cp\u003eThe logD\u003csub\u003e7.4\u003c/sub\u003e of [\u003csup\u003e18\u003c/sup\u003eF\u003cb\u003e]FT-8\u003c/b\u003e was determined in an n-octanol/PBS system. Briefly, [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eFT-8\u003c/b\u003e (100 \u0026micro;L) was mixed with 3 mL 1\u0026times; PBS and 2.9 mL n-octanol, vortexed (3 min), and centrifuged (3000 rpm, 10 min). The n-octanol phase was serially partitioned against fresh PBS. Aliquots (100 \u0026micro;L) from each phase were measured with a WALLAC/Wizard 2480 γ-counter (PerkinElmer, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eProtonation state determination\u003c/h2\u003e \u003cp\u003eThe p\u003cem\u003eK\u003c/em\u003ea values were determined using UV spectroscopic titration on a Sirius T3 instrument (Sirius Analytical Instruments, UK) and analyzed with Sirius T3Dt software (v1.0.12.120) by WuXi AppTec Co., Ltd. (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence staining\u003c/h2\u003e \u003cp\u003ePre-treated slices were incubated with compound \u003cb\u003e23\u003c/b\u003e (1 \u0026micro;M, 10% ethanol) for 5 min at room temperature, rinsed in ethanol, and washed with water (1 min). Fluorescence images were captured on a DMi8 automated microscope imaging system (Leica, Germany) equipped with a Leica K8 (A23B726014) camera and appropriate filter sets.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eAntigen retrieval was performed in preheated buffers (lambda light chain: 0.01 M sodium citrate, pH\u0026thinsp;=\u0026thinsp;6.0; transthyretin: Tris-EDTA, pH\u0026thinsp;=\u0026thinsp;9.0) at 95\u0026deg;C for 15 minutes. Slices were blocked (3% BSA, 2 h) and incubated overnight at 4\u0026deg;C with primary antibody (anti-lambda light chain antibody, ab124719, 1:400 in TBS or anti-transthyretin antibody, 11891-1-AP, 1:100 in TBS). After TBST washes, slices were incubated with secondary antibody (goat anti-rabbit 488 nm, ab150077) at room temperature for 2 h. Nuclei were counterstained with DAPI. Fluorescence images were acquired on a DMi8 automated microscope imaging system (Leica, Germany). Pseudocolors were assigned to lambda light chain (yellow) and transthyretin (green).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCongo Red staining\u003c/h2\u003e \u003cp\u003ePre-treated slices were stained with Congo Red solution (#G1531, Solarbio) for 30 min at room temperature and imaged under polarized light using a DMi8 automated microscope imaging system (Leica, Germany) equipped with a FLEXACAM C3 (1923260073) camera.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMicro-PET/CT imaging in rats.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMale SD rats (6\u0026ndash;10 weeks, male) were anesthetized with a mixture of isoflurane and air (2.5 L/min), and tail intravenously administered [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eFT-3\u003c/b\u003e (7.205 MBq, n\u0026thinsp;=\u0026thinsp;1, 10% ethanol), [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eFT-4\u003c/b\u003e (7.293 MBq, n\u0026thinsp;=\u0026thinsp;1, 10% ethanol), [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eFT-5\u003c/b\u003e (6.013\u0026ndash;6.839 MBq, n\u0026thinsp;=\u0026thinsp;2, 10% ethanol) and [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eFT-8\u003c/b\u003e (7.918\u0026ndash;9.387 MBq, n\u0026thinsp;=\u0026thinsp;2, 10% ethanol). PET/CT included dynamic cardiac imaging (0\u0026ndash;60 min), whole-body PET scanning (60\u0026ndash;70 min), and CT acquisition. Cardiac PET frames: 60 s \u0026times; 15, 300 s \u0026times; 5, 600 s \u0026times; 2. Data were reconstructed by 3D-OSEM algorithm and analyzed by PMOD 4.0 software (PMOD Technologies, Switzerland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003ePET/CT imaging in humans\u003c/h2\u003e \u003cp\u003eOne healthy volunteer (41 years old, 82 kg, male) and one patient with systemic amyloidosis (71 years old, 53 kg, male) underwent [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eFT-8\u003c/b\u003e PET/CT imaging, using a Discovery 710 PET/CT system (GE Healthcare, USA) from the top of the head to mid-thigh. No fasting was required before the scanning. For healthy volunteer, following a low-dose, non-contrast CT acquisition (3.75 mm slice thickness, tube voltage: 120 kV, Scan Type: Helical, Rotation time: 0.8, Rotation Length: Full, Pitch \u0026amp; Speed: 1.375:1 \u0026amp; 55.00), [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eFT-8\u003c/b\u003e (381.1 MBq) was administered intravenously at the bedside. Dynamic PET acquisition commenced immediately and continued for 6 consecutive whole-body scans (1 min/bed; 7 beds), followed by a single whole-body scan (2 min/bed; 7 beds). A second dynamic PET acquisition was performed at 120 min post-injection, consisting of 4 consecutive whole-body scans (2 min/bed; 7 beds). For the patient, PET scan was performed after a same low-dose, non-contrast CT acquisition from the top of the head to mid-thigh at 180 min post-injection (432.9 MBq). All PET data were reconstructed using ordered-subset expectation maximization (2 iterations, 8 subsets) with a Gaussian filter (full width at half maximum 5 mm, image size 168 \u0026times; 168). All reconstructed images were surveyed in axial, coronal, and sagittal planes and analyzed by PMOD 4.1 (PMOD Technologies, Switzerland).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eReporting summary\u003c/h2\u003e\n\u003cp\u003eFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eAll supporting materials are available in the Supplementary Information. Source data are provided with this paper. The raw and analyzed datasets generated during this study are accessible for research purposes from the corresponding author upon reasonable request.\u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eAll authors declare no competing financial interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eM.C. conceptualized the project and designed the methodology. M.C., Y.L., S.Y., J.D., L.Y., D.C., X.Y., and J.Z. contributed materials and funding. J.T., M.W., Q.Z., J.W., and Y.L. conducted all the preclinical experiments. J.T., S.Y., and X.Z. performed radiosynthesis. J.T., M.P., H.Y., and S.Y. carried out first-in-human PET imaging and data analysis. J.T. prepared the original draft, and Y.L., M.C., M.P., H.Y., and S.Y. reviewed and edited the manuscript. All authors approved the final version.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant Nos. 22376016, 22306015, 22022601, and U1967221).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWechalekar, A. D., Gillmore, J. D. \u0026amp; Hawkins, P. N. Systemic amyloidosis. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e387\u003c/strong\u003e, 2641\u0026ndash;2654 (2016).\u003c/li\u003e\n\u003cli\u003eFalk, R. H., Alexander, K. M., Liao, R. \u0026amp; Dorbala, S. AL (light-chain) cardiac amyloidosis. \u003cem\u003eJ. Am. Coll. Cardiol.\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 1323\u0026ndash;1341 (2016).\u003c/li\u003e\n\u003cli\u003eRuberg, F. L., Grogan, M., Hanna, M., Kelly, J. W. \u0026amp; Maurer, M. S. Transthyretin amyloid cardiomyopathy. \u003cem\u003eJ. Am. Coll. 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Pathol.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 413\u0026ndash;417 (2016).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7823866/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7823866/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEarly detection of cardiac amyloidosis (CA) is challenging, despite advances in repurposed \u003cem\u003eβ\u003c/em\u003e-amyloid PET tracers and amyloid-targeted [\u003csup\u003e124\u003c/sup\u003eI]Evuzamitide. The heterogeneity of CA subtypes requires invasive tests, like tissue biopsy, before targeted therapy can begin. Amyloidogenic light chains (AL) expose negatively charged pockets enriched in acidic residues and N-glycosylated modifications, guiding the design of selective molecular probes. We showed that protonation-driven recognition accomplishes unprecedented selectivity for AL deposits. Using this principle, we developed a fluorinated derivative, [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eFT-8\u003c/b\u003e, based on a 4-pyridylpiperazine scaffold, which displayed high binding affinity (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e = 11.52 nM) and selectivity towards AL, as well as favorable pharmacokinetics. In first-in-human PET studies, [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eFT-8\u003c/b\u003e provided high-contrast visualization of AL deposits in the myocardium and extracardiac organs with significant specificity. These findings position [\u003csup\u003e18\u003c/sup\u003eF]\u003cb\u003eFT-8\u003c/b\u003e as a promising PET tracer for advancing the non-invasive differential diagnosis of AL-CA and highlighting the role of protonation in developing new-generation molecular probes across diverse disease contexts.\u003c/p\u003e","manuscriptTitle":"Protonation-Guided Design and Evaluation of Selective PET Tracers for Light Chain Cardiac Amyloidosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-03 15:50:19","doi":"10.21203/rs.3.rs-7823866/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a2cb2323-7e66-4650-b64c-daeca68536ea","owner":[],"postedDate":"February 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57033386,"name":"Biological sciences/Biological techniques/Imaging/Positron-emission tomography"},{"id":57033387,"name":"Health sciences/Cardiology/Cardiovascular biology/Cardiovascular diseases/Cardiomyopathies/Cardiac hypertrophy"},{"id":57033388,"name":"Health sciences/Diseases/Cardiovascular diseases/Cardiomyopathies/Cardiac hypertrophy"},{"id":57033389,"name":"Biological sciences/Biochemistry/Protein folding/Protein aggregation"},{"id":57033390,"name":"Health sciences/Biomarkers/Diagnostic markers"}],"tags":[],"updatedAt":"2026-05-02T14:50:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-03 15:50:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7823866","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7823866","identity":"rs-7823866","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}