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Positron emission tomography is a widely used imaging platform for studying physiological processes. Despite the proliferation of modern synthetic methodologies for radiolabeling, the optimization of these reactions still primarily relies on inefficient one-factor-at-a-time approaches. High-throughput experimentation (HTE) has proven to be a powerful approach for optimizing reactions in many areas of chemical synthesis. However, to date, HTE has rarely been applied to radiochemistry. This is largely because of the short lifetime of common radioisotopes, which presents major challenges for efficient parallel reaction setup and analysis using standard equipment and workflows. Herein, we demonstrate an effective HTE workflow and apply it to the optimization of copper-mediated radiofluorination of pharmaceutically relevant boronate ester substrates. The workflow utilizes commercial equipment and allows for rapid analysis of reactions for optimizing reactions, exploring chemical space using pharmaceutically relevant aryl boronates for radiofluorinations, and constructing large radiochemistry data sets.
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Cobre , Tomografía de Emisión de Positrones , Radioquímica , Tomografía de Emisión de Positrones/métodos , Radiofármacos , Radioisótopos de FlúorRESUMEN
Preclinical models of neurological diseases and gene therapy are essential for neurobiological research. However, the evaluation of such models lacks reliable reporter systems for use with noninvasive imaging methods. Here, we report the development of a reporter system based on the CLIP-tag enzyme and [18F]pFBC, an 18F-labeled covalent CLIP-tag-ligand synthesized via a DoE-optimized and fully automated process. We demonstrated its specificity using a subcutaneous xenograft model and a model of viral vector-mediated brain gene transfer by engineering HEK293 cells and striatal neurons to express membrane-tethered CLIP-tag protein. After in vitro characterization of the reporter, mice carrying either CLIP-tag expressing or control subcutaneous xenografts underwent dynamic [18F]pFBC PET imaging. The CLIP-tag expressing xenografts showed a significantly higher uptake than control xenografts (tumor-to-muscle ratio 5.0 vs 1.7, p = 0.0379). In vivo, metabolite analysis by radio-HPLC from plasma and brain homogenates showed only one radio-metabolite in plasma and none in the brain. In addition, [18F]pFBC showed fast uptake and rapid clearance from the brain in animals injected with adeno-associated virus (AAV)-CLIP in the right striatum but no right-to-left (R-L) uptake difference in the striata in the acquired PET data. In contrast, autoradiography showed a clear accumulation of radioactivity in the AAV-CLIP-injected right striatum compared to the sham-injected left striatum control. CLIP-tag expression and brain integrity were verified by immunofluorescence and light sheet microscopy. In conclusion, we established a novel reporter gene system for PET imaging of gene expression in the brain and periphery and demonstrated its potential for a wide range of applications, particularly for neurobiological research and gene therapy with viral vectors.
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Tomografía de Emisión de Positrones , Radiofármacos , Humanos , Ratones , Animales , Genes Reporteros , Células HEK293 , Radiofármacos/metabolismo , Tomografía de Emisión de Positrones/métodos , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismoRESUMEN
PURPOSE: Tumor hypoxia and other microenvironmental factors are key determinants of treatment resistance. Hypoxia positron emission tomography (PET) and functional magnetic resonance imaging (MRI) are established prognostic imaging modalities to identify radiation resistance in head-and-neck cancer (HNC). The aim of this preclinical study was to develop a multi-parametric imaging parameter specifically for focal radiotherapy (RT) dose escalation using HNC xenografts of different radiation sensitivities. METHODS: A total of eight human HNC xenograft models were implanted into 68 immunodeficient mice. Combined PET/MRI using dynamic [18F]-fluoromisonidazole (FMISO) hypoxia PET, diffusion-weighted (DW), and dynamic contrast-enhanced MRI was carried out before and after fractionated RT (10 × 2 Gy). Imaging data were analyzed on voxel-basis using principal component (PC) analysis for dynamic data and apparent diffusion coefficients (ADCs) for DW-MRI. A data- and hypothesis-driven machine learning model was trained to identify clusters of high-risk subvolumes (HRSs) from multi-dimensional (1-5D) pre-clinical imaging data before and after RT. The stratification potential of each 1D to 5D model with respect to radiation sensitivity was evaluated using Cohen's d-score and compared to classical features such as mean/peak/maximum standardized uptake values (SUVmean/peak/max) and tumor-to-muscle-ratios (TMRpeak/max) as well as minimum/valley/maximum/mean ADC. RESULTS: Complete 5D imaging data were available for 42 animals. The final preclinical model for HRS identification at baseline yielding the highest stratification potential was defined in 3D imaging space based on ADC and two FMISO PCs ([Formula: see text]). In 1D imaging space, only clusters of ADC revealed significant stratification potential ([Formula: see text]). Among all classical features, only ADCvalley showed significant correlation to radiation resistance ([Formula: see text]). After 2 weeks of RT, FMISO_c1 showed significant correlation to radiation resistance ([Formula: see text]). CONCLUSION: A quantitative imaging metric was described in a preclinical study indicating that radiation-resistant subvolumes in HNC may be detected by clusters of ADC and FMISO using combined PET/MRI which are potential targets for future functional image-guided RT dose-painting approaches and require clinical validation.
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Imagen de Difusión por Resonancia Magnética , Neoplasias de Cabeza y Cuello , Humanos , Animales , Ratones , Tomografía de Emisión de Positrones/métodos , Misonidazol , Imagen por Resonancia Magnética , Neoplasias de Cabeza y Cuello/diagnóstico por imagen , Neoplasias de Cabeza y Cuello/radioterapia , Hipoxia , RadiofármacosRESUMEN
A technique to image α-synuclein (αSYN) fibrils in vivo is an unmet scientific and clinical need that would represent a transformative tool in the understanding, diagnosis, and treatment of various neurodegenerative diseases. Several classes of compounds have shown promising results as potential PET tracers, but no candidate has yet exhibited the affinity and selectivity required to reach clinical application. We hypothesized that the application of the rational drug design technique of molecular hybridization to two promising lead scaffolds could enhance the binding to αSYN up to the fulfillment of those requirements. By combining the structures of SIL and MODAG tracers, we developed a library of diarylpyrazoles (DAPs). In vitro evaluation through competition assays against [3H]SIL26 and [3H]MODAG-001 showed the novel hybrid scaffold to have preferential binding affinity for amyloid ß (Aß) over αSYN fibrils. A ring-opening modification on the phenothiazine building block to produce analogs with increased three-dimensional flexibility did not result in an improved αSYN binding but a complete loss of competition, as well as a significant reduction in Aß affinity. The combination of the phenothiazine and the 3,5-diphenylpyrazole scaffolds into DAP hybrids did not generate an enhanced αSYN PET tracer lead compound. Instead, these efforts identified a scaffold for promising Aß ligands that may be relevant to the treatment and monitoring of Alzheimer's disease (AD).
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Humanos , Péptidos beta-Amiloides/metabolismo , alfa-Sinucleína/metabolismo , Enfermedad de Alzheimer/metabolismo , AmiloideRESUMEN
Apicomplexan parasites cause severe disease in both humans and their domesticated animals. Since these parasites readily develop drug resistance, development of new, effective drugs to treat infection caused by these parasites is an ongoing challenge for the medical and veterinary communities. We hypothesized that invertebrate-bacterial symbioses might be a rich source of anti-apicomplexan compounds because invertebrates are susceptible to infections with gregarines, parasites that are ancestral to all apicomplexans. We chose to explore the therapeutic potential of shipworm symbiotic bacteria as they are bona fide symbionts, are easily grown in axenic culture and have genomes rich in secondary metabolite loci [1,2]. Two strains of the shipworm symbiotic bacterium, Teredinibacter turnerae, were screened for activity against Toxoplasma gondii and one strain, T7901, exhibited activity against intracellular stages of the parasite. Bioassay-guided fractionation identified tartrolon E (trtE) as the source of the activity. TrtE has an EC50 of 3 nM against T. gondii, acts directly on the parasite itself and kills the parasites after two hours of treatment. TrtE exhibits nanomolar to picomolar level activity against Cryptosporidium, Plasmodium, Babesia, Theileria, and Sarcocystis; parasites representing all branches of the apicomplexan phylogenetic tree. The compound also proved effective against Cryptosporidium parvum infection in neonatal mice, indicating that trtE may be a potential lead compound for preclinical development. Identification of a promising new compound after such limited screening strongly encourages further mining of invertebrate symbionts for new anti-parasitic therapeutics.
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Antiprotozoarios , Apicomplexa/crecimiento & desarrollo , Bivalvos/microbiología , Gammaproteobacteria/metabolismo , Simbiosis , Animales , Antiprotozoarios/metabolismo , Antiprotozoarios/farmacología , Ratones , Infecciones por Protozoos/tratamiento farmacológicoRESUMEN
BACKGROUND: Postoperative stereotactic radiosurgery (SRS) is a standard management option for patients with resected brain metastases. Preoperative SRS may have certain advantages compared to postoperative SRS, including less uncertainty in delineation of the intact tumor compared to the postoperative resection cavity, reduced rate of leptomeningeal dissemination postoperatively, and a lower risk of radiation necrosis. The recently published ASCO-SNO-ASTRO consensus statement provides no recommendation for the preferred sequencing of radiotherapy and surgery for patients receiving both treatments for their brain metastases. METHODS: This multicenter, randomized controlled trial aims to recruit 88 patients with resectable brain metastases over an estimated three-year period. Patients with ten or fewer brain metastases with at least one resectable, fulfilling inclusion criteria will be randomized to postoperative SRS (standard arm) or preoperative SRS (investigational arm) in a 1:1 ratio. Randomization will be stratified by age (< 60 versus ≥60 years), histology (melanoma/renal cell carcinoma/sarcoma versus other), and number of metastases (one versus 2-10). In the standard arm, postoperative SRS will be delivered within 3 weeks of surgery, and all unresected metastases will receive primary SRS. In the investigational arm, enrolled patients will receive SRS of all brain metastases followed by surgery of resectable metastases within one week of SRS. In either arm, single fraction or hypofractionated SRS in three or five fractions is permitted. The primary endpoint is to assess local control at 12 months in both arms. Secondary endpoints include local control at other time points, regional/distant brain recurrence rates, leptomeningeal recurrence rates, overall survival, neurocognitive outcomes, and adverse radiation events including radiation necrosis rates in both arms. DISCUSSION: This trial addresses the unanswered question of the optimal sequencing of surgery and SRS in the management of patients with resectable brain metastases. No randomized data comparing preoperative and postoperative SRS for patients with brain metastases has been published to date. TRIAL REGISTRATION: Clinicaltrials.gov , NCT04474925; registered on July 17, 2020. Protocol version 1.0 (January 31, 2020). SPONSOR: Alberta Health Services, Edmonton, Canada (Samir Patel, MD).
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Neoplasias Encefálicas , Radiocirugia , Humanos , Persona de Mediana Edad , Radiocirugia/métodos , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/cirugía , Neoplasias Encefálicas/patología , Encéfalo/patología , Necrosis/etiología , Alberta , Resultado del TratamientoRESUMEN
Cryptosporidium sp. are apicomplexan parasites that cause significant morbidity and possible mortality in humans and valuable livestock. There are no drugs on the market that are effective in the population most severely affected by this parasite. This study is the first high-throughput screen for potent anti-Cryptosporidium natural products sourced from a unique marine compound library. The Harbor Branch Oceanographic Institute at Florida Atlantic University has a collection of diverse marine organisms some of which have been subjected to medium pressure liquid chromatography to create an enriched fraction library. Numerous active compounds have been discovered from this library, but it has not been tested against Cryptosporidium parvum. A high-throughput in vitro growth inhibition assay was used to test 3764 fractions in the library, leading to the identification of 23 fractions that potently inhibited the growth of Cryptosporidium parvum. Bioassay guided fractionation of active fractions from a deep-sea sponge, Leiodermatium sp., resulted in the purification of leiodolide A, the major active compound in the organism. Leiodolide A displayed specific anti-Cryptosporidium activity at a half maximal effective concentration of 103.5 nM with selectivity indexes (SI) of 45.1, 11.9, 19.6 and 14.3 for human ileocecal colorectal adenocarcinoma cells (HCT-8), human hepatocellular carcinoma cells (Hep G2), human neuroblastoma cells (SH-SY5Y) and green monkey kidney cells (Vero), respectively. The unique structure of leiodolide A provides a valuable drug scaffold on which to develop new anti-Cryptosporidium compounds and supports the importance of screening natural product libraries for new chemical scaffolds.
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Productos Biológicos , Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animales , Productos Biológicos/farmacología , Línea Celular , Chlorocebus aethiops , Criptosporidiosis/parasitología , Ensayos Analíticos de Alto Rendimiento , HumanosRESUMEN
A convenient and scalable base-free method for processing [18F]fluoride as [18F]TBAF is reported and applied to copper-mediated radiofluorination radiosyntheses. A central feature of this method is that a single production of [18F]TBAF can be divided into small aliquots that can be used to perform multiple small-scale reactions in DoE optimization studies. The results of these studies can then be reliably translated to full batch tracer productions using automated synthesizers. The processing method was applied to the DoE optimization of [18F]olaparib, affording the tracer in high radiochemical yields via both manual (%RCY = 78 ± 6%, n = 4 (CMRF step only)) and automated (up to 80% (%RCY); 41% activity yield (%AY)) radiosynthesis procedures.
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BACKGROUND: Meningiomas are the most common benign intracranial tumors. Gamma Knife® stereotactic radiosurgery (GKSRS) has become a preferred management for recurrent or residual meningiomas. This study focuses on the relationship between tumor control and the time interval between resection of a World Health Organization (WHO) grade 1 meningioma and GKSRS. METHODS: This single institution retrospective analysis reviewed our experience in 238 patients who underwent GKSRS after a pathologically confirmed WHO grade 1 meningioma resection. The median follow-up was 7.4 years. The median aggregate tumor volume at GKSRS was 6 cm3 and a median margin dose of 13 Gy was utilized. Neurological symptoms were evident in 60% of patients at the time of procedure. RESULTS: Overall actuarial tumor control rates achieved were 91.3% at 5 years, 83.4% at 10 years, and 76% at 15 years. There were 35 patients (15%) who developed tumor progression within or directly adjacent to the GKSRS treatment field. The median time until progression was 6.3 years. The duration between surgical intervention and GKSRS did not show statistical significance at 3 months (p = 0.9), 6 months (p = 0.8), 12 months (p = 0.5), or 24 months (p = 0.9). Fifteen patients (6%) had tumor progression at an anatomically distinct location outside the GKSRS target volume. Neurological symptomatic improvement was more likely with early radiosurgery intervention (p = 0.007). CONCLUSION: Postoperative GKSRS was associated with excellent long-term tumor control for WHO grade 1 meningiomas, regardless of the interval after initial surgery. In addition, earlier radiosurgery was associated with superior symptom improvement.
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Neoplasias Meníngeas , Meningioma , Radiocirugia , Niño , Estudios de Seguimiento , Humanos , Neoplasias Meníngeas/radioterapia , Neoplasias Meníngeas/cirugía , Meningioma/radioterapia , Meningioma/cirugía , Radiocirugia/métodos , Estudios Retrospectivos , Resultado del Tratamiento , Carga TumoralRESUMEN
Anosmia is a common outcome after resection of olfactory groove meningioma(s) (OGM) and for some patients represents a significant disability. To evaluate long term tumor control rates and preservation of subjective olfaction after Gamma Knife (GK) stereotactic radiosurgery (SRS) of OGM. We performed a retrospective chart review and telephone assessments of 41 patients who underwent GK SRS between 1987 and 2008. Clinical outcomes were stratified by full, partial or no subjective olfaction, whereas tumor control was assessed by changes in volume greater or lesser than 25%. The median clinical and imaging follow-up were 76 and 65 months, respectively. Prior to SRS, 19 (46%) patients had surgical resections and two (5%) had received fractionated radiation therapy. Twenty four patients (59%) reported a normal sense of smell, 12 (29%) reported a reduced sense of smell and five (12%) had complete anosmia. The median tumor volume was 8.5 cm(3) (range 0.6-56.1), the mean radiation dose at the tumor margin was 13 Gy (range 10-20) and the median estimated dose to the olfactory nerve was 5.1 Gy (range 1.1-18.1). At follow-up, 27 patients (66%) reported intact olfaction (three (7%) described return to a normal sense of smell), nine (22%) described partial anosmia, and five (12%) had complete anosmia. No patient reported deterioration in olfaction after SRS. Thirteen patients (32%) showed significant tumor regression, 26 (63%) had no further growth and two (5%) had progressed. The progression free tumor control rates were 97% at 1 year and 95% at 2, 10 and 20 years. Symptomatic adverse radiation effects occurred in three (7%) patients. Stereotactic radiosurgery provided both long term tumor control and preservation of olfaction.
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Neoplasias Meníngeas/cirugía , Meningioma/cirugía , Radiocirugia/métodos , Olfato/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Estudios de Seguimiento , Humanos , Estimación de Kaplan-Meier , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Dosis de Radiación , Estudios Retrospectivos , Olfato/efectos de la radiación , Tomografía Computarizada por Rayos X , Resultado del Tratamiento , Carga Tumoral/efectos de la radiaciónRESUMEN
This report describes a method for the photochemical Cu-mediated fluorination of aryl iodides with AgF via putative aryl radical (Arâ¢) intermediates. It involves irradiating an aryl iodide with UVB light (λmax = 313 nm) in the presence of a mixture of CuI and CuII salts and AgF. Under these conditions, fluorination proceeds at room temperature for substrates containing diverse substituents, including alkoxy and alkyl groups, ketones, esters, sulfonate esters, sulfonamides, and protected amines. This method has been translated to radiofluorination using a combination of K18F, K3PO4, and AgOTf.
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The development of positron emission tomography (PET) tracers capable of detecting α-synuclein (α-syn) aggregates in vivo would represent a breakthrough for advancing the understanding and enabling the early diagnosis of Parkinson's disease and related disorders. It also holds the potential to assess the efficacy of therapeutic interventions. However, this remains challenging due to different structures of α-syn aggregates, the need for selectivity over other structurally similar amyloid proteins, like amyloid-ß (Aß), which frequently coexist with α-syn pathology, and the low abundance of the target in the brain that requires the development of a high-affinity ligand. To develop a successful PET tracer for the central nervous system (CNS), stringent criteria in terms of polarity and molecular size must also be considered, as the tracer must penetrate the blood-brain barrier and have low nonspecific binding to brain tissue. Here, we report a series of arylpyrazolethiazole (APT) derivatives, rationally designed from a structure-activity relationship study centered on existing ligands for α-syn fibrils, with a particular focus on the selectivity toward α-syn fibrils and control of physicochemical properties suitable for a CNS PET tracer. In vitro competition binding assays performed against [3H]MODAG-001 using recombinant α-syn and Aß1-42 fibrils revealed APT-13 with an inhibition constant of 27.8 ± 9.7 nM and a selectivity of more than 3.3 fold over Aß. Radiolabeled [11C]APT-13 demonstrated excellent brain penetration in healthy mice with a peak standardized uptake value of 1.94 ± 0.29 and fast washout from the brain (t 1/2 = 9 ± 1 min). This study highlights the potential of APT-13 as a lead compound for developing PET tracers to detect α-syn aggregates in vivo.
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We present a photo- and Cu-mediated 11C cyanation of bench-stable (hetero)aryl thianthrenium salts via an aryl radical addition pathway. The thianthrenium substrates can be readily accessed via C-H functionalization, and the radiocyanation protocol proceeds under mild conditions (<50 °C, 5 min) and can be automated using open-source, readily accessible augmentations to existing radiochemistry equipment.
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Recent Food and Drug Administration (FDA) approval of diagnostic and therapeutic radiopharmaceuticals and concurrent miniaturization of particle accelerators leading to improved access has fueled interest in the development of chemical transformations suitable for short-lived radioactive isotopes on the tracer scale. This recent renaissance of radiochemistry is paired with new opportunities to study fundamental chemical behavior and reactivity of elements to improve their production, separation, and incorporation into bioactive molecules to generate new radiopharmaceuticals. This outlook outlines pertinent challenges in the field of radiochemistry and indicates areas of opportunity for chemical discovery and development, including those of clinically established (C-11, F-18) and experimental radionuclides in preclinical development across the periodic table.
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Prolactin (PRL) has recently been demonstrated to elicit female-selective nociceptor sensitization and increase pain-like behaviors in female animals. Here we report the discovery and characterization of first-in-class, humanized PRL neutralizing monoclonal antibodies (PRL mAbs). We obtained two potent and selective PRL mAbs, PL 200,031 and PL 200,039. PL 200,031 was engineered as human IgG1 whereas PL 200,039 was reformatted as human IgG4. Both mAbs have sub-nanomolar affinity for human PRL (hPRL) and produce concentration-dependent and complete inhibition of hPRL signaling at the hPRL receptor (hPRLR). These two PRL mAbs are selective for hPRL as they do not inhibit other hPRLR agonists such as human growth hormone or placental lactogen. They also cross-react with non-human primate PRL but not with rodent PRL. Further, both mAbs show long clearance half-lives after intravenous administration in FcRn-humanized mice. Consistent with their isotypes, these mAbs only differ in binding affinities to Fcγ receptors, as expected by design. Finally, PL 200,019, the murine parental mAb of PL 200,031 and PL 200,039, fully blocked stress-induced and PRL-dependent pain behaviors in female PRL-humanized mice, thereby providing in vivo preclinical proof-of-efficacy for PRL mAbs in mechanisms relevant to pain in females.
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Prolactina , Receptores de Prolactina , Femenino , Ratones , Animales , Embarazo , Prolactina/metabolismo , Prolactina/farmacología , Receptores de Prolactina/metabolismo , Anticuerpos Monoclonales , Placenta/metabolismo , Unión ProteicaRESUMEN
A method to detect and quantify aggregated α-synuclein (αSYN) fibrils in vivo would drastically impact the current understanding of multiple neurodegenerative diseases, revolutionizing their diagnosis and treatment. Several efforts have produced promising scaffolds, but a notable challenge has hampered the establishment of a clinically successful αSYN positron emission tomography (PET) tracer: the requirement of high selectivity over the other misfolded proteins amyloid ß (Aß) and tau. By designing and screening a library of 2-styrylbenzothiazoles based on the selective fluorescent probe RB1, this study aimed at developing a selective αSYN PET tracer. [3H]PiB competition binding assays identified PFSB (Ki = 25.4 ± 2.3 nM) and its less lipophilic analogue MFSB, which exhibited enhanced affinity to αSYN (Ki = 10.3 ± 4.7 nM) and preserved selectivity over Aß. The two lead compounds were labeled with fluorine-18 and evaluated using in vitro autoradiography on human brain slices, where they demonstrated up to 4-fold increased specific binding in MSA cases compared to the corresponding control, reasonably reflecting selective binding to αSYN pathology. In vivo PET imaging showed [18F]MFSB successfully crosses the blood-brain barrier (BBB) and is taken up in the brain (SUV = 1.79 ± 0.02). Although its pharmacokinetic profile raises the need for additional structural optimization, [18F]MFSB represents a critical step forward in the development of a successful αSYN PET tracer by overcoming the major challenge of αSYN/Aß selectivity.
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The staging and local management of breast cancer involves the evaluation of the extent and completeness of excision of both the invasive carcinoma component and also the intraductal component or ductal carcinoma in situ. When both invasive ductal carcinoma and coincident ductal carcinoma in situ are present, assessment of the extent and localization of both components is required for optimal therapeutic planning. We have used a mouse model of breast cancer to evaluate the feasibility of applying molecular imaging to assess the local status of cancers in vivo. Multi-tracer positron emission tomography (PET) and magnetic resonance imaging (MRI) characterize the transition from premalignancy to invasive carcinoma. PET tracers for glucose consumption, membrane synthesis, and neoangiogenesis in combination with a Gaussian mixture model-based analysis reveal image-derived thresholds to separate the different stages within the whole-lesion. Autoradiography, histology, and quantitative image analysis of immunohistochemistry further corroborate our in vivo findings. Finally, clinical data further support our conclusions and demonstrate translational potential. In summary, this preclinical model provides a platform for characterizing multistep tumor progression and provides proof of concept that supports the utilization of advanced protocols for PET/MRI in clinical breast cancer imaging.
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There is a need for versatile in vivo nuclear imaging reporter systems to foster preclinical and clinical research. We explore the applicability of the SNAPTag and novel radiolabeled small-molecule ligands as a versatile reporter gene system for in vivo nuclear imaging. SNAPTag is a high-affinity protein tag used in a variety of biochemical research areas and based on the suicide DNA repair enzyme O6-methylguanine methyl transferase (MGMT). Its ligands are well suited for reporter gene imaging as the benzyl guanine core scaffold can be derivatized with fluorescent or radiolabeled moieties for various applications. Three guanine-based SNAPTag ligands ([18F]FBBG, [18F]pFBG and [18F]mFBG) were synthesized in high yields and were (radio)chemically characterized. HEK293 cells were engineered to express the SNAPTag on the cell surface and served as cell model to assess target affinity by radiotracer uptake assays, Western blotting and SDS-PAGE autoradiography. A subcutaneous HEK293-SNAPTag xenograft model in immunodeficient mice was used for in vivo evaluation of [18F]FBBG and [18F]pFBG while the biodistribution of [18F]mFBG was characterized in naïve animals. The results were validated by ex vivo biodistribution studies and immunofluorescence staining of the xenografts. All three radiotracers were produced in high radiochemical purity, molar activity and good yields. Western blot analysis revealed successful SNAPTag expression by the transfected HEK293 cells. In vitro testing revealed high target affinity of all three tracers with an up to 191-fold higher signal in the HEK293-SNAPTag cells compared to untransfected cells. This was further supported by a prominent radioactive protein band at the expected size in the SDS-PAGE autoradiograph of cells incubated with [18F]FBBG or [18F]pFBG. The in vivo studies demonstrated high uptake in HEK293-SNAP xenografts compared to HEK293 xenografts with excellent tumor-to-muscle ratios (7.5 ± 4.2 for [18F]FBBG and 10.6 ± 6.2 for [18F]pFBG). In contrast to [18F]pFBG and its chemical analogue [18F]mFBG, [18F]FBBG showed no signs of unspecific bone uptake and defluorination in vivo. Radiolabeled SNAPTag ligands bear great potential for clinical applications such as in vivo tracking of cell populations, antibody fragments and targeted radiotherapy. With excellent target affinity, good stability, and low non-specific binding, [18F]FBBG is a highly promising candidate for further preclinical evaluation.
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Given the clinical potential of poly(ADP-ribose) polymerases (PARP) imaging for the detection and stratification of various cancers, the development of novel PARP imaging probes with improved pharmacological profiles over established PARP imaging agents is warranted. Here, we present a novel 18F-labeled PARP radiotracer based on the clinically superior PARP inhibitor talazoparib. An automated radiosynthesis of [18F]talazoparib (RCY: 13 ± 3.4%; n = 4) was achieved using a "design of experiments" (DoE) optimized copper-mediated radiofluorination reaction. The chiral product was isolated from the reaction mixture using 2D reversed-phase/chiral radio-HPLC (>99% ee). (8S,9R)-[18F]Talazoparib demonstrated PARP binding in HCC1937 cells in vitro and showed an excellent tumor-to-blood ratio in xenograft-bearing mice (10.2 ± 1.5). Additionally, a favorable pharmacological profile in terms of excretion, metabolism, and target engagement was observed. This synthesis of [18F]talazoparib exemplifies how DoE can enable the radiosyntheses of synthetically challenging radiolabeled compounds of high interest to the imaging community.
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Antineoplásicos/farmacología , Automatización , Neoplasias de la Mama/tratamiento farmacológico , Ftalazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Antineoplásicos/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Radioisótopos de Flúor , Humanos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones Endogámicos NOD , Estructura Molecular , Ftalazinas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/análisis , Células Tumorales CultivadasRESUMEN
Neurodegenerative diseases such as Parkinson's disease (PD) are manifested by inclusion bodies of alpha-synuclein (α-syn) also called α-synucleinopathies. Detection of these inclusions is thus far only possible by histological examination of postmortem brain tissue. The possibility of non-invasively detecting α-syn will therefore provide valuable insights into the disease progression of α-synucleinopathies. In particular, α-syn imaging can quantify changes in monomeric, oligomeric, and fibrillic α-syn over time and improve early diagnosis of various α-synucleinopathies or monitor treatment progress. Positron emission tomography (PET) is a non-invasive in vivo imaging technique that can quantify target expression and drug occupancies when a suitable tracer exists. As such, novel α-syn PET tracers are highly sought after. The development of an α-syn PET tracer faces several challenges. For example, the low abundance of α-syn within the brain necessitates the development of a high-affinity ligand. Moreover, α-syn depositions are, in contrast to amyloid proteins, predominantly localized intracellularly, limiting their accessibility. Furthermore, another challenge is the ligand selectivity over structurally similar amyloids such as amyloid-beta or tau, which are often co-localized with α-syn pathology. The lack of a defined crystal structure of α-syn has also hindered rational drug and tracer design efforts. Our objective for this review is to provide a comprehensive overview of current efforts in the development of selective α-syn PET tracers.