Search Results (38)

A novel brain positron emission tomography (PET) radioligand, [¹¹C]HY-2-15, has potential for imaging alpha-synuclein aggregations in multiple system atrophy and misfolded tau proteins in tauopathies, based on its high binding affinity in disease brain tissue homogenates. Here, we demonstrate that [³H]HY-2-15 has the capability to bind to aggregated alpha-synuclein in multiple system atrophy brain and tau aggregations in progressive supranuclear palsy and corticobasal degeneration brain tissues via in vitro autoradiography study. A first-in-human pilot multicenter clinical study recruited a total of 10 subjects including healthy controls and patients with Parkinson’s disease, multiple system atrophy, or progressive supranuclear palsy. The study revealed that [¹¹C]HY-2-15 has a relatively higher specific uptake in the pallidum and midbrain of patients with progressive supranuclear palsy. Total-body scans performed on the PennPET Explorer showed the radiotracer was cleared by renal excretion. However, the rapid metabolism and low brain uptake resulted in a limited signal of [¹¹C]HY-2-15 in brain. Full article

The molecular imaging of α-synuclein (α-syn) pathology in Parkinson’s disease (PD) and related movement disorders is a clinically unmet need. The aim of this study was to discover and characterize in vitro a radioligand for imaging α-syn pathology. A library of 78 small molecules was developed and screened using recombinant α-syn fibrils and brain homogenates from Alzheimer’s disease (AD) donors. The selection criteria were as follows: Ki_α-syn < 30 nM, Ki_tau and Ki_A-β > 200 nM. Three compounds, GMC-073 (K_iα-syn: 8 nM), GMC-098 (Ki_α-syn: 9.7 nM), and GMC-058 (Ki_α-syn: 22.5 nM), fulfilled the criteria and were radiolabeled with ³H. [³H]GMC-058 was the only compound with negligible binding in controls, and was further evaluated using tissue microarrays, autoradiography on fresh-frozen brain slices, and in vitro saturation binding assay on brain homogenates. [³H]GMC-058 binding co-localized with α-syn inclusions in Parkinson’s disease (PD) and multiple-system atrophy (MSA), with dense A-β plaques in cerebral amyloid angiopathy and AD and with p-tau inclusions in progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Specific binding was highest in PSP and CBD. In vitro K_D was highest in AD (5.4 nM), followed by PSP (41 nM) and CBD (75 nM). The K_D in MSA, PD, and controls was >100 nM. [³H]GMC-058 is a novel radioligand displaying a low affinity for aggregated α-syn in tissue, with an in vitro profile also suitable for detecting tau pathology in 4R tauopathies. Full article

There are two types of noninvasive biomarkers of human embryo developmental potential: those based on a direct assessment of embryo morphology over time and those using spent media after embryo in vitro culture as source of information. Both are derived from previously acquired knowledge on different aspects of pre-implantation embryo development. These aspects include embryo morphology and kinetics, chromosomal ploidy status, metabolism, and embryonic gene transcription, translation, and expression. As to the direct assessment of morphology and kinetics, pertinent data can be obtained by analyzing sequential microscopic images of in vitro cultured embryos. Spent media can serve a source of genomic, metabolomic, transcriptomic and proteomic markers. Methods used in the early pioneering studies, such as microscopy, fluorescence in situ hybridization, autoradiography, electrophoresis and immunoblotting, or enzyme-linked immunosorbent assay, are too subjective, invasive, and/or time-consuming. As such, they are unsuitable for the current in vitro fertilization (IVF) practice, which needs objective, rapid, and noninvasive selection of the best embryo for uterine transfer or cryopreservation. This has been made possible by the use of high-throughput techniques such as time-lapse (for direct embryo evaluation), next-generation sequencing, quantitative real-time polymerase chain reaction, high-performance liquid chromatography, nanoparticle tracking analysis, flow cytometry, mass spectroscopy, Raman spectroscopy, near-infrared spectroscopy, and nuclear magnetic resonance spectroscopy (for spent culture media analysis). In this review, individual markers are presented systematically, with each marker’s history and current status, including available methodologies, strengths, and limitations, so as to make the essential information accessible to all health professionals, even those whose expertise in the matter is limited. Full article

Background/Objectives: Fibroblast activation protein (FAP) has gained tremendous traction as a target for tumor imaging and cancer treatment, while also playing a key role in fibrosis. Our study aimed to evaluate [⁶⁸Ga]Ga-DATA^5m.SA.FAPi for PET imaging of replacement fibrosis following myocardial infarction (MI) or interstitial fibrosis associated with hypertrophy. Methods: MI or transverse aortic constriction (TAC)-induced hypertrophy was induced in C57BL/6 mice, with sham-operated animals serving as controls. At multiple time points during disease progression (1, 2, and 6 weeks post-surgery), [⁶⁸Ga]Ga-DATA^5m.SA.FAPi PET/CT scans were performed, followed by ex vivo investigations. Additionally, in vitro cell uptake experiments simulating hypertrophy were conducted. Results: Cardiac uptake of [⁶⁸Ga]Ga-DATA5m.SA.FAPi significantly increased two weeks after MI induction (MI: 2.1 ± 0.2%ID/g, n = 7 vs. SHAM: 1.1 ± 0.2%ID/g, n = 5; p = 0.002), confirmed by ex vivo autoradiography. No significant difference was observed at six weeks post-MI (MI: 1.1 ± 0.1%ID/g, n = 4 vs. SHAM: 0.8 ± 0.0%ID/g, n = 3), indicating infarct healing completion. In contrast, TAC mice showed increased uptake after six weeks (TAC: 1.8 ± 0.2%ID/g, n = 6; p = 0.007), related to interstitial fibrosis progression. Consistently, high-stretched cardiac fibroblasts demonstrated a higher uptake compared to low-stretched conditioned ones, suggesting the stretch mediates regulation of FAP. Conclusions: This study demonstrated the efficacy of [⁶⁸Ga]Ga-DATA^5m.SA.FAPi for longitudinal imaging of cardiac fibrosis in response to different cardiac injuries. In vivo FAP imaging during cardiac remodeling may serve as a valuable tool for diagnosing and predicting disease progression, ultimately aiding in the clinical management of patients. Full article

Background: Bromodomain and extra-terminal (BET) proteins are critical regulators of gene transcription, as they recognize acetylated lysine residues. The BD1 bromodomain of BRD4, a member of the BET family, has emerged as a promising therapeutic target for various diseases. This study aimed to develop and evaluate a novel C-11 labeled PET radiotracer, [¹¹C]YL10, for imaging the BD1 bromodomain of BRD4 in vivo. Methods: [¹¹C]YL10 was synthesized and evaluated for its ability to bind to the BD1 bromodomain selectively. PET imaging studies were conducted in mice to assess brain penetration, pharmacokinetics, and selectivity. In vitro autoradiography and blocking experiments were performed to confirm the tracer’s specificity for the BD1 domain. Results: [¹¹C]YL10 demonstrated good brain penetration, high selectivity for the BD1 bromodomain, and favorable pharmacokinetics in initial PET imaging studies. In vitro autoradiography and blocking experiments confirmed the specific binding of [¹¹C]YL10 to the BD1 domain of BRD4, further validating its potential as a targeted radiotracer. Conclusions: The development of [¹¹C]YL10 provides a new tool for studying BRD4 bromodomains using PET imaging technology. This radiotracer offers potential advancement in the diagnosis and research of neurodegenerative diseases and related disorders involving BRD4 dysregulation. Full article

About 75% of breast tumors show an overexpression of the estradiol receptor (ER), making it a valuable target for tumor diagnosis and therapy. To date, 16α-[¹⁸F]fluoroestradiol (FES) is the only FDA-approved imaging probe for the positron emission tomography (PET) imaging of ER-positive (ER⁺⁾ breast cancer. However, FES has the drawback of a high retention in the liver. Therefore, the aim of this study was the development and preclinical evaluation of estradiol (E2) derivatives with different lipophilicity. Three ¹⁸F-labeled prosthetic groups (two glycosyl and one PEG azide) were chosen for conjugation with ethinyl estradiol (EE) by ¹⁸F-CuAAC (Cu-catalyzed azide-alkyne cycloaddition). The cellular uptake in ER⁺ MCF-7 tumor cells was highest for the less hydrophilic derivative (¹⁸F-TA-Glyco-EE). In nude mice bearing different breast tumors (ER⁺ MCF-7 and T47D versus ER⁻ MDA-MB-231), ¹⁸F-TA-Glyco-EE revealed a high uptake in the liver (13%ID/g, 30 min p.i.), which decreased over 90 min to 1.2%ID/g, indicating fast hepatobiliary clearance. The statistically significant difference of ¹⁸F-TA-Glyco-EE uptake in T47D compared to MDA-MB-231 tumors at 60–90 min p.i. indicated ER-specific uptake, whereas in vivo PET imaging did not provide evidence for specific uptake of ¹⁸F-TA-Glyco-EE in MCF-7 tumors, probably due to ER occupation by E2 after E2-dependent MCF-7 tumor growth in mice. However, in vitro autoradiography revealed a high specific binding of ¹⁸F-TA-Glyco-EE to ER⁺ tumor slices. We conclude that ¹⁸F-TA-Glyco-EE, with its increased hydrophilicity after deacetylation in the blood and thus rapid washout from non-target tissues, may be a viable alternative to FES for the PET imaging of breast cancer. Full article

The synthesis, biochemical evaluation and radiosynthesis of a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor and radioligand was performed. NT431, a newly synthesized 4-fluorobenzyl-abemaciclib, exhibited high potency to CDK4/6 and against four cancer cell lines with IC₅₀ similar to that of the parent abemaciclib. We performed a two-step one-pot radiosynthesis to produce [¹⁸F]NT431 with good radiochemical yield (9.6 ± 3%, n = 3, decay uncorrected), high radiochemical purity (>95%), and high molar activity (>370 GBq/µmol (>10.0 Ci/µmol). In vitro autoradiography confirmed the specific binding of [¹⁸F]NT431 to CDK4/6 in brain tissues. Dynamic PET imaging supports that both [¹⁸F]NT431 and the parent abemaciclib crossed the BBB albeit with modest brain uptake. Therefore, we conclude that it is unlikely that NT431 or abemaciclib (FDA approved drug) can accumulate in the brain in sufficient concentrations to be potentially effective against breast cancer brain metastases or brain cancers. However, despite the modest BBB penetration, [¹⁸F]NT431 represents an important step towards the development and evaluation of a new generation of CDK4/6 inhibitors with superior BBB penetration for the treatment and visualization of CDK4/6 positive tumors in the CNS. Also, [¹⁸F]NT431 may have potential application in peripheral tumors such as breast cancer and other CDK4/6 positive tumors. Full article

Tozadenant (4-hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide) is a highly selective adenosine A_2A receptor (A_2AR) antagonist and a promising lead structure for the development of A_2AR-selective positron emission tomography (PET) probes. Although several ¹⁸F-labelled tozadenant derivatives showed favorable in vitro properties, recent in vivo PET studies observed poor brain penetration and lower specific binding than anticipated from the in vitro data. While these findings might be attributable to the structural modification associated with ¹⁸F-labelling, they could also reflect inherent properties of the parent compound. However, PET studies with radioisotopologues of tozadenant to evaluate its cerebral pharmacokinetics and brain distribution are still lacking. In the present work, we applied N-Boc-O-desmethyltozadenant as a suitable precursor for the preparation of [O-methyl-¹¹C]tozadenant ([¹¹C]tozadenant) by O-methylation with [¹¹C]methyl iodide followed by acidic deprotection. This approach afforded [¹¹C]tozadenant in radiochemical yields of 18 ± 2%, with molar activities of 50–60 GBq/µmol (1300–1600 mCi/µmol) and radiochemical purities of 95 ± 3%. In addition, in vitro autoradiography in pig and rat brain slices demonstrated the expected striatal accumulation pattern and confirmed the A_2AR specificity of the radioligand, making it a promising tool for in vivo PET studies on the cerebral pharmacokinetics and brain distribution of tozadenant. Full article

Aggregation of Tau protein into paired helical filaments causing neurofibrillary tangles (NFT) is a neuropathological feature in Alzheimer’s disease (AD). This study aimed to develop and evaluate the effectiveness of a novel radioiodinated tracer, 4-[¹²⁵I]iodo-3-(1H-pyrrolo[2,3-c]pyridine-1-yl)pyridine ([¹²⁵I]INFT), for binding to Tau protein in postmortem human AD brain. Radiosynthesis of [¹²⁵I]INFT was carried out using electrophilic destannylation by iodine-125 and purified chromatographically. Computational modeling of INFT binding on Tau fibril was compared with IPPI. In vitro, autoradiography studies were conducted with [¹²⁵I]INFT for Tau in AD and cognitively normal (CN) brains. [¹²⁵I]INFT was produced in >95% purity. Molecular modeling of INFT revealed comparable binding energies to IPPI at site-1 of the Tau fibril with an affinity of IC₅₀ = 7.3 × 10⁻⁸ M. Binding of [¹²⁵I]INFT correlated with the presence of Tau in the AD brain, confirmed by anti-Tau immunohistochemistry. The ratio of average grey matter (GM) [¹²⁵I]INFT in AD versus CN was found to be 5.9, and AD GM/white matter (WM) = 2.5. Specifically bound [¹²⁵I]INFT to Tau in AD brains was displaced by IPPI (>90%). Monoamine oxidase inhibitor deprenyl had no effect and clorgyline had little effect on [¹²⁵I]INFT binding. [¹²⁵I]INFT is a less lipophilic imaging agent for Tau in AD. Full article

Pretargeting is a promising nuclear imaging technique that allows for the usage of antibodies (Abs) with enhanced imaging contrast and reduced patient radiation burden. It is based on bioorthogonal chemistry with the tetrazine ligation—a reaction between trans-cyclooctenes (TCOs) and tetrazines (Tzs)—currently being the most popular reaction due to its high selectivity and reactivity. As Abs can be designed to bind specifically to currently ‘undruggable’ targets such as protein isoforms or oligomers, which play a crucial role in neurodegenerative diseases, pretargeted imaging beyond the BBB is highly sought after, but has not been achieved yet. A challenge in this respect is that large molecules such as Abs show poor brain uptake. Uptake can be increased by receptor mediated transcytosis; however, it is largely unknown if the achieved brain concentrations are sufficient for pretargeted imaging. In this study, we investigated whether the required concentrations are feasible to reach. As a model Ab, we used the bispecific anti-amyloid beta (Aβ) anti-transferrin receptor (TfR) Ab 3D6scFv8D3 and conjugated it to a different amount of TCOs per Ab and tested different concentrations in vitro. With this model in hand, we estimated the minimum required TCO concentration to achieve a suitable contrast between the high and low binding regions. The estimation was carried out using pretargeted autoradiography on brain sections of an Alzheimer’s disease mouse model. Biodistribution studies in wild-type (WT) mice were used to correlate how different TCO/Ab ratios alter the brain uptake. Pretargeted autoradiography showed that increasing the number of TCOs as well as increasing the TCO-Ab concentration increased the imaging contrast. A minimum brain concentration of TCOs for pretargeting purposes was determined to be 10.7 pmol/g in vitro. Biodistribution studies in WT mice showed a brain uptake of 1.1% ID/g using TCO-3D6scFv8D3 with 6.8 TCO/Ab. According to our estimations using the optimal parameters, pretargeted imaging beyond the BBB is not a utopia. Necessary brain TCO concentrations can be reached and are in the same order of magnitude as required to achieve sufficient contrast. This work gives a first estimate that pretargeted imaging is indeed possible with antibodies. This could allow the imaging of currently ‘undruggable’ targets and therefore be crucial to monitor (e.g., therapies for intractable neurodegenerative diseases). Full article

The efficacy of thrombolysis is inversely correlated with thrombus age. During early thrombogenesis, activated factor XIII (FXIIIa) cross-links $\alpha$2-AP to fibrin to protect it from early lysis. This was exploited to develop an $\alpha$2-AP-based imaging agent to detect early clot formation likely susceptible to thrombolysis treatment. In this study, this imaging probe was improved and validated using ${}^{111}$In SPECT/CT in a mouse thrombosis model. In vitro fluorescent- and ${}^{111}$In-labelled imaging probe-to-fibrin cross-linking assays were performed. Thrombus formation was induced in C57Bl/6 mice by endothelial damage (FeCl${}_3$) or by ligation (stenosis) of the infrarenal vena cava (IVC). Two or six hours post-surgery, mice were injected with ${}^{111}$In-DTPA-A16 and ExiTron Nano 12000, and binding of the imaging tracer to thrombi was assessed by SPECT/CT. Subsequently, ex vivo IVCs were subjected to autoradiography and histochemical analysis for platelets and fibrin. Efficient in vitro cross-linking of A16 imaging probe to fibrin was obtained. In vivo IVC thrombosis models yielded stable platelet-rich thrombi with FeCl${}_3$ and fibrin and red cell-rich thrombi with stenosis. In the stenosis model, clot formation in the vena cava corresponded with a SPECT hotspot using an A16 imaging probe as a molecular tracer. The fibrin-targeting A16 probe showed specific binding to mouse thrombi in in vitro assays and the in vivo DVT model. The use of specific and covalent fibrin-binding probes might enable the clinical non-invasive imaging of early and active thrombosis. Full article

The 18 kDa translocator protein (TSPO) is increasingly recognized as an interesting target for the imaging of glioblastoma (GBM). Here, we investigated TSPO PET imaging and autoradiography in the frequently used GL261 glioblastoma mouse model and aimed to generate insights into the temporal evolution of TSPO radioligand uptake in glioblastoma in a preclinical setting. We performed a longitudinal [¹⁸F]GE-180 PET imaging study from day 4 to 14 post inoculation in the orthotopic syngeneic GL261 GBM mouse model (n = 21 GBM mice, n = 3 sham mice). Contrast-enhanced computed tomography (CT) was performed at the day of the final PET scan (±1 day). [¹⁸F]GE-180 autoradiography was performed on day 7, 11 and 14 (ex vivo: n = 13 GBM mice, n = 1 sham mouse; in vitro: n = 21 GBM mice; n = 2 sham mice). Brain sections were also used for hematoxylin and eosin (H&E) staining and TSPO immunohistochemistry. [¹⁸F]GE-180 uptake in PET was elevated at the site of inoculation in GBM mice as compared to sham mice at day 11 and later (at day 14, TBR_max +27% compared to sham mice, p = 0.001). In GBM mice, [¹⁸F]GE-180 uptake continuously increased over time, e.g., at day 11, mean TBR_max +16% compared to day 4, p = 0.011. [¹⁸F]GE-180 uptake as depicted by PET was in all mice co-localized with contrast-enhancement in CT and tissue-based findings. [¹⁸F]GE-180 ex vivo and in vitro autoradiography showed highly congruent tracer distribution (r = 0.99, n = 13, p < 0.001). In conclusion, [¹⁸F]GE-180 PET imaging facilitates non-invasive in vivo monitoring of TSPO expression in the GL261 GBM mouse model. [¹⁸F]GE-180 in vitro autoradiography is a convenient surrogate for ex vivo autoradiography, allowing for straightforward identification of suitable models and scan time-points on previously generated tissue sections. Full article

We report [¹⁸F]nifene binding to α4β2* nicotinic acetylcholinergic receptors (nAChRs) in Parkinson’s disease (PD). The study used transgenic Hualpha-Syn(A53T) PD mouse model of α-synucleinopathy for PET/CT studies in vivo and autoradiography in vitro. Additionally, postmortem human PD brain sections comprising of anterior cingulate were used in vitro to assess translation to human studies. Because the small size of mice brain poses challenges for PET imaging, improved methods for radiosynthesis of [¹⁸F]nifene and simplified PET/CT procedures in mice were developed by comparing intravenous (IV) and intraperitoneal (IP) administered [¹⁸F]nifene. An optimal PET/CT imaging time of 30–60 min post injection of [¹⁸F]nifene was established to provide thalamus to cerebellum ratio of 2.5 (with IV) and 2 (with IP). Transgenic Hualpha-Syn(A53T) mice brain slices exhibited 20–35% decrease while in vivo a 20–30% decrease of [¹⁸F]nifene was observed. Lewy bodies and α-synuclein aggregates were confirmed in human PD brain sections which lowered the [¹⁸F]nifene binding by more than 50% in anterior cingulate. Thus [¹⁸F]nifene offers a valuable tool for PET imaging studies of PD. Full article

There is an unmet medical need for non-invasive, sensitive, and quantitative methods for the assessment of fibrosis. Herein, an improved collagelin analogue labelled with gallium-68 for use with positron emission tomography (PET) is presented. A cyclic peptide, c[CPGRVNleHGLHLGDDEGPC], was synthesized by solid-phase peptide synthesis, conjugated to 2-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)acetic acid, and labelled with gallium-68. High performance liquid chromatography (HPLC) was used for the quality and stability assessment of the collagelin analogue. Non-specific organ distribution, blood clearance, and excretion rates were investigated in healthy mice and rats using ex vivo organ distribution analysis and dynamic in vivo PET/CT. Mice with carbon tetrachloride (CCl₄) induced liver fibrosis were used for the investigation of specific binding via in vitro frozen section autoradiography, ex vivo organ distribution, and in vivo PET/CT. A non-decay corrected radiochemical yield (48 ± 6%) of [⁶⁸Ga]Ga-NOTA-PEG₂-c[CPGRVNleHGLHLGDDEGPC] ([⁶⁸Ga]Ga-NO2A-[Nle¹³]-Col) with a radiochemical purity of 98 ± 2% was achieved without radical scavengers. The ⁶⁸Ga-labelling was regioselective and stable at ambient temperature for at least 3 h. The autoradiography of the cryosections of fibrotic mouse liver tissue demonstrated a distinct heterogeneous radioactivity uptake that correlated with the fibrosis scores estimated after Sirius Red staining. The blood clearance and tissue washout from the [⁶⁸Ga]Ga-NO2A-[Nle¹³]-Col was fast in both normal and diseased mice. Dosimetry investigation in rats indicated the possibility for 4–5 PET/CT examinations per year. Radiolytic stability of the collagelin analogue was achieved by the substitution of methionine with norleucine amino acid residue without a deterioration of its binding capability. [⁶⁸Ga]Ga-NO2A-[Nle¹³]-Col demonstrated a safe dosimetry profile suitable for repeated scanning. Full article

In this work, we designed, developed, characterized, and investigated a new chelator and its bifunctional derivative for ⁸⁹Zr labeling and PET-imaging. In a preliminary study, we synthesized two hexadentate chelators named AAZTHAS and AAZTHAG, based on the seven-membered heterocycle AMPED (6-amino-6-methylperhydro-1,4-diazepine) with the aim to increase the rigidity of the ⁸⁹Zr complex by using N-methyl-N-(hydroxy)succinamide or N-methyl-N-(hydroxy)glutaramide pendant arms attached to the cyclic structure. N-methylhydroxamate groups are the donor groups chosen to efficiently coordinate ⁸⁹Zr. After in vitro stability tests, we selected the chelator with longer arms, AAZTHAG, as the best complexing agent for ⁸⁹Zr presenting a stability of 86.4 ± 5.5% in human serum (HS) for at least 72 h. Small animal PET/CT static scans acquired at different time points (up to 24 h) and ex vivo organ distribution studies were then carried out in healthy nude mice (n = 3) to investigate the stability and biodistribution in vivo of this new ⁸⁹Zr-based complex. High stability in vivo, with low accumulation of free ⁸⁹Zr in bones and kidneys, was measured. Furthermore, an activated ester functionalized version of AAZTHAG was synthesized to allow the conjugation with biomolecules such as antibodies. The bifunctional chelator was then conjugated to the human anti-HER2 monoclonal antibody Trastuzumab (Tz) as a proof of principle test of conjugation to biologically active molecules. The final ⁸⁹Zr labeled compound was characterized via radio-HPLC and SDS-PAGE followed by autoradiography, and its stability in different solutions was assessed for at least 4 days. Full article