An Improved HPLC Separation Method for TSPO Radioligand [11C]ER176 Clinical Production.

Mitochondrial membrane translocator protein 18 kDa (TSPO) expression is increased in activated microglia, established as a plausible target of neuroinflammation imaging. [11C]ER176, specifically binding to TSPO, has been developed as the third generation of radioligand for PET imaging of TSPO, which showed the potential in better quantifying neuroinflammation than its predecessors. In the current study, we developed an automated radiosynthesis with an improved HPLC purification method for [11C]ER176 clinical production. The improved HPLC separation was integrated into the automated production of [11C]ER176 using a reverse phase semi-preparative HPLC column with an isocratic pump and the mixture of methanol and 50 mM ammonium acetate as the mobile phase. The fraction corresponding to [11C]ER176 was collected around 8.5-9.0 min without the risk of getting contaminations from nearby impurities. The automated production process took about 30 min after end of bombardment (EOB) and the quality of the final product [11C]ER176 met all specifications for clinical use based on current US Pharmacopeia and FDA CGMP requirements.

Patient-derived tumor xenograft study with CDK4/6 inhibitor plus AKT inhibitor for the management of metastatic castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is an aggressive malignancy with poor outcomes. To investigate novel therapeutic strategies, we characterized three new metastatic prostate cancer PDTX models and developed 3D spheroids from each to investigate molecular targeted therapy combinations including CDK4/6 inhibitors (CDK4/6i) with AKT inhibitors (ATKi). Metastatic prostate cancer tissue was collected and three PDTX models were established and characterized using WES. PDTX 3-D spheroids were developed from these three PDTXs to show resistance patterns and test novel molecular targeted therapies. CDK4/6i's were combined with AKTi's to assess synergistic antitumor response to prove our hypothesis that blockade of AKT overcomes drug resistance to CDK4/6 inhibitor. This combination was evaluated in PDTX 3-D spheroids and in vivo experiments with responses measured by tumor volumes, PSA and Ga-68 PSMA-11 PET-CT imaging. We demonstrated CDK4/6i's with AKTi's possess synergistic antitumor activity in three mCRPC PDTX models. These models have multiple unique pathogenic and deleterious genomic alterations with resistance to single agent CDK4/6i's. Despite this, combination therapy with AKTi's was able to overcome resistance mechanisms. The IHC and Western blot analysis confirmed on target effects, while tumor volume, serum PSA ELISA, and radionuclide imaging demonstrated response to therapy with statistically significant SUV differences seen with Ga-68 PSMA-11 PET-CT. These preclinical data demonstrating antitumor synergy by overcoming single agent CDK 4/6i as well as AKTi drug resistance provide the rational for a clinical trial combining a CDK4/6i with an AKTi in mCRPC patients whose tumor expresses wild type RB1.

Radiosynthesis and Preliminary Evaluation of [11C]SSI-4 for the Positron Emission Tomography Imaging of Stearoyl CoA Desaturase 1.

Stearoyl CoA desaturase 1 (SCD1) is the rate-limiting enzyme for converting saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs) and plays a key role in endogenous (de novo) fatty acid metabolism. Given that this pathway is broadly upregulated across many tumor types with an aggressive phenotype, SCD1 has emerged as a compelling target for cancer imaging and therapy. The ligand 2-(4-(2-chlorophenoxy)piperidine-1-carboxamido)-N-methylisonicotinamide (SSI-4) was identified as a potent and highly specific SCD1 inhibitor with a strong binding affinity for SCD1 at our laboratory. We herein report the radiosynthesis of [11C]SSI-4 and the preliminary biological evaluation including in vivo PET imaging of SCD1 in a human tumor xenograft model. Radiotracer [11C]SSI-4 was labeled at the carbamide position via the direct [11C]CO2 fixation on the Synthra MeIplus module in high molar activity and good radiochemical yield. In vitro cell uptake assays were performed with three hepatocellular carcinoma (HCC) cell lines and three renal cell carcinoma (RCC) cell lines. Additionally, in vivo small animal PET/CT imaging with [11C]SSI-4 and the biodistribution were carried out in a mouse model bearing HCC xenografts. Radiotracer [11C]SSI-4 afforded a 4.14 ± 0.44% (decay uncorrected, n = 10) radiochemical yield based on starting [11]CO2 radioactivity. The [11C]SSI-4 radiosynthesis time including HPLC purification and SPE formulation was 25 min from the end of bombardment to the end of synthesis (EOS). The radiochemical purity of [11C]SSI-4 was 98.45 ± 1.43% (n = 10) with a molar activity of 225.82 ± 33.54 GBq/µmol (6.10 ± 0.91 Ci/µmol) at the EOS. In vitro cell uptake study indicated all SSI-4 responsive HCC and RCC cell line uptakes demonstrate specific uptake and are blocked by standard compound SSI-4. Preliminary small animal PET/CT imaging study showed high specific uptake and block of [11C]SSI-4 uptake with co-injection of cold SSI-4 in high SCD1-expressing organs including lacrimal gland, brown fat, liver, and tumor. In summary, novel radiotracer [11C]SSI-4 was rapidly and automatedly radiosynthesized by direct [11C]CO2 fixation. Our preliminary biological evaluation results suggest [11C]SSI-4 could be a promising radiotracer for PET imaging of SCD1 overexpressing tumor tissues.

Automated radiosynthesis of 1-(2-[18 F]fluoroethyl)-L-tryptophan ([18 F]FETrp) for positron emission tomography (PET) imaging of cancer in humans.

The radiotracer 1-(2-[18 F]fluoroethyl)-L-tryptophan (L-[18 F]FETrp or [18 F]FETrp) is a substrate of indoleamine 2,3-dioxygenase, the initial and key enzyme of the kynurenine pathway associated with tumoral immune resistance. In preclinical positron emission tomography studies, [18 F]FETrp is highly accumulated in a wide range of primary and metastatic cancers, such as lung cancer, prostate cancer, and gliomas. However, the clinical translation of this radiotracer into the first-in-human trial has not been reported, partially due to its racemization during radiofluorination which renders the purification of the final product challenging. However, efficient purification is essential for human studies in order to assure radiochemical and enantiomeric purity. In this work, we report a fully automated radiosynthesis of [18 F]FETrp on a Synthra RNPlus research module, including a one-pot two steps radiosynthesis, dual independent chiral and reverse-phase semipreparative high-performance liquid chromatography purifications, and solid-phase extraction-assisted formulation. The presented approach has led to its Investigational New Drug application and approval that allows the testing of this tracer in humans.

The Continuum of Metastatic Prostate Cancer: Interpreting PSMA PET Findings in Recurrent Prostate Cancer.

Conventional imaging has been the standard imaging modality for assessing prostate cancer recurrence and is utilized to determine treatment response to therapy. Molecular imaging with PSMA PET-CT has proven to be more accurate, sensitive, and specific at identifying pelvic or distant metastatic disease, resulting in earlier diagnosis of advanced disease. Since advanced disease may not be seen on conventional imaging, due to its lower sensitivity, but can be identified by molecular imaging, this reveals that metastatic prostate cancer occurs on a continuum from negative PSMA PET-CT and negative conventional imaging to positive PSMA PET-CT and positive conventional imaging. Understanding this continuum, the accuracy of these modalities, and treatment related outcomes based on imaging, will allow the clinician to counsel patients on management. This review will highlight the differences in conventional and molecular imaging in prostate cancer and how PSMA PET-CT can be used for the management of prostate cancer patients in different clinical scenarios, while providing cautionary notes for overtreatment.

Sparstolonin B suppresses free fatty acid palmitate-induced chondrocyte inflammation and mitigates post-traumatic arthritis in obese mice.

Abnormal lipid metabolism, such as systemic increased free fatty acid, results in overproduction of pro-inflammatory enzymes and cytokines, which is crucial in the development of obesity-related osteoarthritis (OA). However, there are only a few drugs that target the lipotoxicity of OA. Recent researches have documented that the traditional Chinese medicine, Sparstolonin B (Ssn B), exerted anti-inflammatory effects in various diseases, but not yet in OA. On the basis of this evidence, our works purposed to evaluate the effect of Ssn B on free fatty acid (FFA) palmitate (PA)-stimulated human osteoarthritic chondrocytes and obesity-associated mouse OA model. We found that Ssn B suppressed PA-triggered inflammatory response and extracellular matrix catabolism in a concentration-dependent approach. In vivo, Ssn B treatment inhibited cartilage degeneration and subchondral bone calcification caused by joint mechanical imbalance and alleviated metabolic inflammation in obesity. Mechanistically, co-immunoprecipitine and molecular docking analysis showed that the formation of toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) complex caused by PA was blocked by Ssn B. Subsequently, it leads to inactivation of PA-caused myeloid differentiation factor 88 (MyD88)-dependent nuclear factor-kappaB (NF-κB) cascade. Together, these findings demonstrated that Ssn B is a potential treatment agent for joint degenerative diseases in obese individuals.

HPLC-free and cassette-based nucleophilic production of [18F]FDOPA for clinical use.

Radiotracer 3,4-dihydroxy-6-[18F]fluoro-L-phenylalanine (L-6-[18F]fluorodopa or [18F]FDOPA) is widely used for PET imaging of dopamine metabolism in several diseases including Parkinson's Disease, brain tumor, neuroendocrine tumors, and focal hyperinsulinism of infancy. In 2019, [18F]FDOPA was approved by US FDA for detection of dopaminergic nerve terminals in the striatum of adult patients with suspected Parkinsonian Syndromes. A convenient and reliable method is desired for fully automated production of [18F]FDOPA under cGMP compliance to meet the increasing clinical need. In this study, we reported a cassette-based automated production of [18F]FDOPA using a GE Fastlab 2 module and the quality control (QC) under fully cGMP compliant environment. Briefly, automated radiosynthesis of [18F]FDOPA was processed via nucleophilic radio-fluorination using FDOPA Fastlab cassette and solid phase extraction (SPE) purification. The QC tests of [18F]FDOPA, including appearance, pH, half-life, radiochemical purity and identity, enantiomeric purity, chemical impurities, molecular activity, radioactive concentration, filter integrity, endotoxin, and sterility, were conducted at the end of synthesis (EOS) and 8 h after EOS during the validation runs. Three consecutive productions of [18F]FDOPA were reliably achieved with desired radiochemical yield and high radiochemical/enantiomeric purities and molar activity. The uncorrected radiochemical yields of [18F]FDOPA were 9.3-9.8% with a total synthesis time of ~140 min. Both radiochemical and enantiomeric purities of [18F]FDOPA were >99.9% and the molar activities were 2.1-3.9 Ci/µmole at EOS. The full QC results at EOS and 8 h after EOS showed that the produced [18F]FDOPA met all release criteria for clinical use within 8 hours of expiration time. Three consecutive validation runs and QC results demonstrated the efficacy of cassette-based production of [18F]FDOPA for routine clinical use.

Automated production of [18F]Flortaucipir for PET imaging of tauopathies.

Radiotracer [18F]Flortaucipir is an FDA-approved diagnostic agent for PET imaging of density and distribution of abnormal tau protein deposition (tauopathies) in Alzheimer's disease. A high-yield automated method for routine GMP-compliant [18F]Flortaucipir production is desired to meet increasing clinical need. In this work, we reported an automated radiosynthesis of [18F]Flortaucipir in a RNplus Research module and the quality control (QC) tests for human use under full GMP compliance. Briefly, automated radiosynthesis of [18F]Flortaucipir was processed via nucleophilic radiofluorination of precursor AV1622 and followed by acid hydrolysis in a RNplus Research module, which included the radiosynthesis, semi-preparative high-performance liquid chromatography (HPLC) purification, and the final formulation via solid phase extraction (SPE). The final products were obtained in non-decay corrected radiochemical yields of 14.8-16.6% (n = 3) within total synthesis time of 55 min. The radiochemical purities of [18F]Flortaucipir were > 99.9% and the molar activities were 247.9-384.8 GBq/µmol at end of synthesis. The results of QC tests met all the specifications for human use. In conclusion, [18F]Flortaucipir was reproducibly achieved with desired radiochemical yield and high radiochemical purity and molar activity. Three GMP compliant validation runs and QC results demonstrated the efficacy of this method for automated production of [18F]Flortaucipir for human use.

Analysis of [11C]Choline and [13N]Ammonia using a single HPIC method.

A single HPIC method was developed and validated for the analysis of both [11C]Choline and [13N]Ammonia with the same setup. The HPIC system suitability tests were performed and [11C]Choline and [13N]Ammonia were used to verify their performance on this HPIC method. The HPIC setup and method provides qualitative and quantitative analysis information of [11C]Choline and [13N]Ammonia. The data suggested this HPIC method is validated for determining radiochemical/chemical purity and radiochemical identity of [11C]Choline and [13N]Ammonia products in CGMP compliant manufacturing process.

PET imaging of medulloblastoma with an 18F-labeled tryptophan analogue in a transgenic mouse model.

In vivo positron emission tomography (PET) imaging is a key modality to evaluate disease status of brain tumors. In recent years, tremendous efforts have been made in developing PET imaging methods for pediatric brain tumors. Carbon-11 labelled tryptophan derivatives are feasible as PET imaging probes in brain tumor patients with activation of the kynurenine pathway, but the short half-life of carbon-11 limits its application. Using a transgenic mouse model for the sonic hedgehog (Shh) subgroup of medulloblastoma, here we evaluated the potential of the newly developed 1-(2-[18F]fluoroethyl)-L-tryptophan (1-L-[18F]FETrp) as a PET imaging probe for this common malignant pediatric brain tumor. 1-L-[18F]FETrp was synthesized on a PETCHEM automatic synthesizer with good chemical and radiochemical purities and enantiomeric excess values. Imaging was performed in tumor-bearing Smo/Smo medulloblastoma mice with constitutive actvation of the Smoothened (Smo) receptor using a PerkinElmer G4 PET-X-Ray scanner. Medulloblastoma showed significant and specific accumulation of 1-L-[18F]FETrp. 1-L-[18F]FETrp also showed significantly higher tumor uptake than its D-enantiomer, 1-D-[18F]FETrp. The uptake of 1-L-[18F]FETrp in the normal brain tissue was low, suggesting that 1-L-[18F]FETrp may prove a valuable PET imaging probe for the Shh subgroup of medulloblastoma and possibly other pediatric and adult brain tumors.

Evaluation of L-1-[18F]Fluoroethyl-Tryptophan for PET Imaging of Cancer.

PURPOSE: Fluorine-18 labeled tryptophan analog L-1-[18F]fluoroethyl-tryptophan (L-1-[18F]FETrp) was designed for positron emission tomography (PET) imaging of cancer by dual targeting of the overexpressed amino acid transporters and altered indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway of tryptophan metabolism. In our previous study, we described the radiosynthesis and preliminary evaluation of L-1-[18F]FETrp for PET imaging of breast cancer. The aim of this study was to investigate the in vivo imaging mechanism and further evaluate this radiotracer in more wide range types of cancers including prostate cancer, lung cancer, and glioma. PROCEDURES: The mice bearing subcutaneous PC-3 prostate cancer, subcutaneous H2009 and H460 lung cancers, subcutaneous MDA-MB-231, orthotopic A549 lung cancer, and intracranial 73C glioma were employed to evaluate L-1-[18F]FETrp for PET imaging of cancer. The in vivo catabolism of L-1-[18F]FETrp in the tumor was studied by analysis of PC-3 extracts with radio-HPLC. RESULTS: Small animal PET/CT imaging of L-1-[18F]FETrp visualized all tumors in these different mouse models with high accumulations of radioactivity in PC-3 (7.5 ± 0.6 % ID/g), H2009 (5.3 ± 0.8 % ID/g), H460 (9.0 ± 1.4 % ID/g), A549 (4.5 ± 0.5 % ID/g), and 73C (4.1 ± 0.7 % ID/g) tumors. The radio-HPLC analysis of PC-3 tumor extracts revealed that about 30 % of L-1-[18F]FETrp was converted into a highly polar radioactive metabolite. The uptake in H460 cancer was about 1.7-fold higher than that in H2009 cancer, which indicated L-1-[18F]FETrp could differentiate these subtypes of lung cancers (H2009 and H460) by imaging quantification. Furthermore, small animal PET/CT imaging in intracranial glioma revealed L-1-[18F]FETrp could pass blood-brain barrier (BBB) and accumulate in glioma with a favorable imaging contrast (tumor-to-brain 2.9). CONCLUSIONS: L-1-[18F]FETrp highly accumulated in a wide range of malignancies including lung cancer, prostate cancer, and glioma. These results suggested that L-1-[18F]FETrp is a promising radiotracer for PET imaging of cancer.

Assessment of Tryptophan Uptake and Kinetics Using 1-(2-18F-Fluoroethyl)-l-Tryptophan and α-11C-Methyl-l-Tryptophan PET Imaging in Mice Implanted with Patient-Derived Brain Tumor Xenografts.

Abnormal tryptophan metabolism via the kynurenine pathway is involved in the pathophysiology of a variety of human diseases including cancers. α-11C-methyl-l-tryptophan (11C-AMT) PET imaging demonstrated increased tryptophan uptake and trapping in epileptic foci and brain tumors, but the short half-life of 11C limits its widespread clinical application. Recent in vitro studies suggested that the novel radiotracer 1-(2-18F-fluoroethyl)-l-tryptophan (18F-FETrp) may be useful to assess tryptophan metabolism via the kynurenine pathway. In this study, we tested in vivo organ and tumor uptake and kinetics of 18F-FETrp in patient-derived xenograft mouse models and compared them with 11C-AMT uptake. METHODS: Xenograft mouse models of glioblastoma and metastatic brain tumors (from lung and breast cancer) were developed by subcutaneous implantation of patient tumor fragments. Dynamic PET scans with 18F-FETrp and 11C-AMT were obtained for mice bearing human brain tumors 1-7 d apart. The biodistribution and tumoral SUVs for both tracers were compared. RESULTS: 18F-FETrp showed prominent uptake in the pancreas and no bone uptake, whereas 11C-AMT showed higher uptake in the kidneys. Both tracers showed uptake in the xenograft tumors, with a plateau of approximately 30 min after injection; however, 18F-FETrp showed higher tumoral SUV than 11C-AMT in all 3 tumor types tested. The radiation dosimetry for 18F-FETrp determined from the mouse data compared favorably with the clinical 18F-FDG PET tracer. CONCLUSION: 18F-FETrp tumoral uptake, biodistribution, and radiation dosimetry data provide strong preclinical evidence that this new radiotracer warrants further studies that may lead to a broadly applicable molecular imaging tool to examine abnormal tryptophan metabolism in human tumors.

Improved Radiosynthesis and Biological Evaluations of L- and D-1-[18F]Fluoroethyl-Tryptophan for PET Imaging of IDO-Mediated Kynurenine Pathway of Tryptophan Metabolism.

PURPOSE: Tryptophan metabolism via indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway plays a role in immunomodulation and has been emerging as a plausible target for cancer immunotherapy. Imaging IDO-mediated kynurenine pathway of tryptophan metabolism with positron emission tomography (PET) could provide valuable information for noninvasive assessment of cancer immunotherapy response. In this work, radiotracer 1-(2-[18F]fluoroethyl)-L-tryptophan (1-L-[18F]FETrp) and its enantioisomer 1-D-[18F]FETrp were synthesized and evaluated for PET imaging of IDO-mediated kynurenine pathway of tryptophan metabolism. PROCEDURES: Enantiopure 1-L-[18F]FETrp and 1-D-[18F]FETrp were prepared by a nucleophilic reaction of N-boc-1-(2-tosylethyl) tryptophan tert-butyl ester with [18F]Fluoride, followed by acid hydrolysis in a GE Tracerlab FX-N module. In vitro cell uptake assays were performed with a breast cancer cell line MDA-MB-231. Small animal PET/computed tomography (CT) imaging was carried out in a mouse model bearing MDA-MB-231 xenografts. RESULTS: Automatic radiosynthesis of 1-L-[18F]FETrp and 1-D-[18F]FETrp was achieved by a one-pot two-step approach in 19.0 ± 7.0 and 9.0 ± 3.0 % (n = 3) decay-corrected yield with radiochemical purity over 99 %, respectively. In vitro cell uptake study indicated the uptake of 1-D-[18F]FETrp in MDA-MB-231 cells was 0.73 ± 0.07 %/mg of protein at 60 min, while, the corresponding uptake of 1-L-[18F]FETrp was 6.60 ± 0.77 %/mg. Further mechanistic assays revealed that amino acid transport systems L-tpye amino acid transporter (LAT) and alanine-, serine-, and cysteine-preferring (ASC), and enzyme IDO expression were involved in cell uptake of 1-L-[18F]FETrp. Small animal PET/CT imaging study showed the tumor uptake of 1-L-[18F]FETrp was 4.6 ± 0.4 % ID/g, while, the tumor uptake of 1-D-[18F]FETrp was low to 1.0 ± 0.2 % ID/g, which were confirmed by ex vivo biodistribution study. CONCLUSIONS: We have developed a practical method for the automatic radiosynthesis of 1-L-[18F]FETrp and 1-D-[18F]FETrp. Our biological evaluation results suggest that 1-L-[18F]FETrp is a promising radiotracer for PET imaging of IDO-mediated kynurenine pathway of tryptophan metabolism in cancer.

Design, radiosynthesis, and evaluation of radiotracers for positron emission tomography imaging of stearoyl-CoA desaturase-1.

Design, radiosynthesis, and biological evaluation of two radiotracers (N-(3-[(18)F]fluoropropyl)-6-(4-(trifluoromethyl)benzoyl)-piperazin-1-yl)pyridazine-3-carboxamide ((18)F-FPPPT) and (N-(4-[(18)F]fluoroaniline)-6-(4-(trifluoromethyl)benzoyl)-piperazin-1-yl)pyridazine-3-carboxamide ((18)F-FAPPT)) are described for noninvasive assessment of stearoyl-CoA desaturase-1 (SCD-1). The overexpression of SCD-1 in multiple solid tumors associates with poor survival in cancer patients. The two radiotracers, (18)F-FPPPT and (18)F-FAPPT, were each prepared in three steps in radiochemical yields of 21% and 3%, respectively. The practicality of imaging SCD-1 with (18)F-FPPPT was tested in two mouse models bearing xenograft tumors with different levels of SCD-1 expression, which afforded a 1.8-fold uptake difference correspondingly. Our work indicates that it is possible to develop SCD-1 specific imaging probes from previously reported SCD-1 inhibitors.

Radiosynthesis of (11)C-Levetiracetam: A Potential Marker for PET Imaging of SV2A Expression.

The multistep preparation of (11)C-levetiracetam ((11)C-LEV) was carried out by a one-pot radiosynthesis with 8.3 ± 1.6% (n = 8) radiochemical yield in 50 ± 5.0 min. Briefly, the propionaldehyde was converted to propan-1-imine in situ as labeling precursor by incubation with ammonia. Without further separation, the imine was reacted with (11)C-HCN to form (11)C-aminonitrile. This crude was then reacted with 4-chlorobutyryl chloride and followed by hydrolysis to yield (11)C-LEV after purification by chiral high-performance liquid chromatography (HPLC). Both the radiochemical and enantiomeric purities of (11)C-LEV were >98%.

RGD-based PET tracers for imaging receptor integrin αv ß3 expression.

Positron emission tomography (PET) imaging of receptor integrin αv ß3 expression may play a key role in the early detection of cancer and cardiovascular diseases, monitoring disease progression, evaluating therapeutic response, and aiding anti-angiogenic drugs discovery and development. The last decade has seen the development of new PET tracers for in vivo imaging of integrin αv ß3 expression along with advances in PET chemistry. In this review, we will focus on the radiochemistry development of PET tracers based on arginine-glycine-aspartic acid (RGD) peptide, present an overview of general strategies for preparing RGD-based PET tracers, and review the recent advances in preparations of (18) F-labeled, (64) Cu-labeled, and (68) Ga-labeled RGD tracers, RGD-based PET multivalent probes, and RGD-based PET multimodality probes for imaging receptor integrin αv ß3 expression.

Fully automated production of 11C-doxepin for PET imaging histamine H1 receptor.

OBJECTIVES: (11)C-Doxepin is an established positron emission tomography (PET) probe for imaging the histamine H1 receptor, which is associated with various neurological disorders and allergic diseases. A fully automated current Good Manufacturing Practices (cGMP)-compliant radiosynthesis is therefore desirable in order to facilitate clinical PET studies. We report here a fully automated production method for (11)C-doxepin using a multipurpose PET module for clinical use. METHODS: (11)C-Doxepin was radiosynthesized by N-[(11)C]methylation of nordoxepin using [(11)C]methyl iodide in DMF solvent, and then purified by HPLC, and finally reformulated with solid phase extraction (SPE) using a cGMP-compliant automated multipurpose PET module developed in house. The final product was analyzed and subjected to quality control according to current US Pharmacopeia requirements. RESULTS: The radiochemical yield (decay corrected) of (11)C-doxepin for clinical use was 47.0 ± 5.2% (n = 12) based on [(11)C]methyl iodide, moreover the radiochemical purity of (11)C-doxepin was more than 97.5% with 1,200 ± 500 Ci/mmol specific activity(end of production). The total production time of (11)C-doxepin was 37 min from end of bombardment (EOB) with the final product passing all tests under cGMP requirements for clinical use. CONCLUSIONS: A simplified and reliable fully automated production of (11) C-doxepin for clinical use was developed, allowing the synthesis of the tracer with high yield using a cGMP-compliant module and procedure. The success of this approach could make the PET tracer (11) C-doxepin more accessible for clinical studies.

Improved synthesis of 2'-deoxy-2'-[18F]fluoro-5-methyl-1-ß-D-arabinofuranosyluracil ([18F]FMAU).

[18F]FMAU is an established PET probe used to monitor cellular proliferation. For clinical applications, a fully automated cGMP-compliant radiosynthesis would be preferred. However, the current synthesis of [18F]FMAU requires HBr activation of the sugar prior to the coupling with silylated uracil. This multiple step procedure makes the development of an automated protocol difficult and complicated. In this study, we report the use of Friedel-Crafts catalysts for an improved synthesis of [18F]FMAU, which also includes a significantly simplified one-pot reaction condition.

(64)Cu labeled AmBaSar-RGD2 for micro-PET imaging of integrin αvß3 expression.

Integrin αvß3 plays a critical role in tumor-induced angiogenesis and metastasis. Previously, a 64Cu-AmBaSar- RGD monomer with high in vivo stability compared with 64Cu-DOTA-RGD was developed for integrin αvß3 PET imaging. It has been established that dimeric RGD peptides have higher receptor-binding affinity and superior in vivo kinetics compared with monomeric RGD peptides due to the polyvalency effect. In this context, we synthesized and evaluated 64Cu-labeled AmBaSar dimeric RGD conjugates (64Cu-AmBaSar-RGD2) for PET imaging of integrin αvß3 expression. The dimeric RGD peptide was conjugated with a cage-like chelator AmBaSar and labeled with 64Cu. Cell binding, microPET imaging, receptor blocking, and biodistribution studies of 64Cu-AmBaSar-RGD2 were conducted in the U87MG human glioblastoma xenograft model. AmBaSar-RGD2 conjugate was obtained in reasonable yield (45.0 ± 2.5%, n= 4) and the identity was confirmed by HPLC and MS (found 1779.8, calculated m/z for [M+H]+ M: C81H125N27O19 1779.9). 64Cu-AmBaSar-RGD2 was obtained with high radiochemical yield (92.0 ± 1.3%) and purity (≥ 98.0%) under mild conditions (pH 5.0~5.5, 23~37 °C) in 30 min. The specific activity of 64Cu-AmBaSar-RGD2 was estimated to be 15-22 GBq/µmol at the end of synthesis. Based on microPET imaging and biodistribution studies, 64Cu-AmBaSar-RGD2 has demonstrated higher tumor uptake at selected time points than 64Cu-AmBaSar-RGD. At 20 h p.i., the tumor uptake reached 0.65 ± 0.05 %ID/g for 64Cu-AmBaSar-RGD and 1.76 ± 0.38 %ID/g for 64Cu-AmBaSar-RGD2, respectively. The integrin αvß3 targeting specificity was confirmed by blocking experiments. Therefore, the new tracer 64Cu-AmBaSar- RGD2 exhibited better tumor-targeting efficacy and more favorable in vivo pharmacokinetics than the 64Cu labeled RGD monomer due to the polyvalency effect.

The improved syntheses of 5-substituted 2'-[18F]fluoro-2'-deoxy-arabinofuranosyluracil derivatives ([18F]FAU, [18F]FEAU, [18F]FFAU, [18F]FCAU, [18F]FBAU and [18F]FIAU) using a multistep one-pot strategy.

INTRODUCTION: We and others have previously reported a four-step radiosynthesis of a series of 2'-deoxy-2'-[(18)F]fluoro-5-substituted-1-ß-D-arabinofuranosyluracil derivatives including [(18)F]FAU, [(18)F]FEAU, [(18)F]FFAU, [(18)F]FCAU, [(18)F]FBAU and [(18)F]FIAU as thymidine derivatives for tumor proliferation and/or reporter gene expression imaging with positron emission tomography (PET). Although the radiosynthesis has been proven to be reproducible and efficient, this complicated multistep reaction is difficult to incorporate into an automated cGMP-compliant radiosynthesis module for routine production. Recently, we have developed a simple and efficient one-pot method for routine production of [(18)F]FMAU. In this study, we studied the feasibility of radiosynthesizing [(18)F]FAU, [(18)F]FEAU, [(18)F]FFAU, [(18)F]FCAU, [(18)F]FBAU and [(18)F]FIAU using this newly developed method. METHODS: Similar to the radiosynthesis of [(18)F]FMAU, 5-substituted 2'-[(18)F]fluoro-2'-deoxy-arabinofuranosyluracil derivatives ([(18)F]FAU, [(18)F]FEAU, [(18)F]FFAU, [(18)F]FCAU, [(18)F]FBAU and [(18)F]FIAU) were synthesized in one-pot radiosynthesis module in the presence of Friedel-Crafts catalyst TMSOTf and HMDS. RESULTS: This one-pot radiosynthesis method could be used to produce [(18)F]FAU, [(18)F]FEAU, [(18)F]FFAU, [(18)F]FCAU, [(18)F]FBAU and [(18)F]FIAU. The overall radiochemical yields of these tracers varied from 4.1%±0.8% to 10.1%±1.9% (decay-corrected, n=4). The overall reaction time was reduced from 210 min to 150 min from the end of bombardment, and the radiochemical purity was >99%. CONCLUSIONS: The improved radiosyntheses of [(18)F]FAU, [(18)F]FEAU, [(18)F]FFAU, [(18)F]FCAU, [(18)F]FBAU and [(18)F]FIAU have been achieved with reasonable yields and high purity using a multistep one-pot method. The synthetic time has been reduced, and the reaction procedures have been significantly simplified. The success of this approach may make PET tracers [(18)F]FAU, [(18)F]FEAU, [(18)F]FFAU, [(18)F]FCAU, [(18)F]FBAU and [(18)F]FIAU more accessible for preclinical and clinical research.