Improved GMP-compliant multi-dose production and quality control of 6-[18F]fluoro-L-DOPA

Background: 6-[18F]Fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) is a frequently used radiopharmaceutical for detecting neuroendocrine and brain tumors and for the differential diagnosis of Parkinson’s disease. To meet the demand for FDOPA, a high-yield GMP-compliant production method is required. Therefore, this study aimed to improve the FDOPA production and quality control procedures to enable distribution of the radiopharmaceutical over distances. FDOPA was prepared by electrophilic fluorination of the trimethylstannyl precursor with [18F]F2, produced from [18O]2 via the double-shoot approach, leading to FDOPA with higher specific activity as compared to FDOPA which was synthesized, using [18F]F2 produced from 20Ne, leading to FDOPA with a lower specific activity. The quality control of the product was performed using a validated UPLC system and compared with quality control with a conventional HPLC system. Impurities were identified using UPLC-MS. Results: The [18O]2 double-shoot radionuclide production method yielded significantly more [18F]F2 with less carrier F2 than the conventional method starting from 20Ne. After adjustment of radiolabeling parameters substantially higher amounts of FDOPA with higher specific activity could be obtained. Quality control by UPLC was much faster and detected more side-products than HPLC. UPLC-MS showed that the most important side-product was FDOPA-quinone, rather than 6-hydroxydopa as suggested by the European Pharmacopoeia. Conclusion: The production and quality control of FDOPA were significantly improved by introducing the [18O]2 double-shoot radionuclide production method, and product analysis by UPLC, respectively. As a result, FDOPA is now routinely available for clinical practice and for distribution over distances

GMP Compliant Synthesis of Canagliflozin, a Novel PET Tracer for the Sodium−Glucose Cotransporter 2

[Image: see text] Inhibition of the sodium–glucose cotransporter 2 (SGLT2) by canagliflozin in type 2 diabetes mellitus results in large between-patient variability in clinical response. To better understand this variability, the positron emission tomography (PET) tracer [(18)F]canagliflozin was developed via a Cu-mediated (18)F-fluorination of its boronic ester precursor with a radiochemical yield of 2.0 ± 1.9% and a purity of >95%. The GMP automated synthesis originated [(18)F]canagliflozin with a yield of 0.5–3% (n = 4) and a purity of >95%. Autoradiography showed [(18)F]canagliflozin binding in human kidney sections containing SGLT2. Since [(18)F]canagliflozin is the isotopologue of the extensively characterized drug canagliflozin and thus shares its toxicological and pharmacological characteristics, it enables its immediate use in patients

Clinical-grade N-(4-[18F]fluorobenzoyl)-interleukin-2 for PET imaging of activated T-cells in humans

BACKGROUND: Molecular imaging of immune cells might be a potential tool for response prediction, treatment evaluation and patient selection in inflammatory diseases as well as oncology. Targeting interleukin-2 (IL2) receptors on activated T-cells using positron emission tomography (PET) with N-(4-[18F]fluorobenzoyl)-interleukin-2 ([18F]FB-IL2) could be such a strategy. This paper describes the challenging translation of the partly manual labeling of [18F]FB-IL2 for preclinical studies into an automated procedure following Good Manufacturing Practices (GMP), resulting in a radiopharmaceutical suitable for clinical use. METHODS: The preclinical synthesis of [18F]FB-IL2 was the starting point for translation to a clinical production method. To overcome several challenges, major adaptations in the production process were executed. The final analytical methods and production method were validated and documented. All data with regards to the quality and safety of the final drug product were documented in an investigational medicinal product dossier. RESULTS: Restrictions in the [18F]FB-IL2 production were imposed by hardware configuration of the automated synthesis equipment and by use of disposable cassettes. Critical steps in the [18F]FB-IL2 production comprised the purification method, stability of recombinant human IL2 and the final formulation. With the GMP compliant production method, [18F]FB-IL2 could reliably be produced with consistent quality complying to all specifications. CONCLUSIONS: To enable the use of [18F]FB-IL2 in clinical studies, a fully automated GMP compliant production process was developed. [18F]FB-IL2 is now produced consistently for use in clinical studies

Pharmacokinetic properties of radiolabeled mutant Interleukin-2v:a PET imaging study

Interleukin-2 (IL2) is a cytokine that can stimulate cytotoxic immune cells to attack infected and malignant cells. Unfortunately, IL2 can also cause serious immune-related toxicity. Recently, a mutant of IL2 (IL2v) with abolished CD25 binding, increased plasma half-life and less toxicity was engineered. Unlike wild-type IL2 (wt-IL2), mutant IL2v does not bind to the α-subunit (CD25) of the high affinity IL2αβγ receptor, but only to its β and γ subunit. Here, we investigated the biological properties of IL2v and compared with the wt-IL2 using fluorine-18 and PET. [18F]FB-IL2v binds specifically to IL2 receptors (IL2R) on activated human peripheral blood monocytes (hPBMCs) and is cleared mainly by the kidneys (Balb/c mice). [18F]FB-IL2v PET studies in SCID mice injected with hPBMCs revealed high uptake in the implant (0.85 ± 0.15 SUV), which was significantly reduced after pretreatment with wt-IL2 or mutant IL2v (SUV 0.26 ± 0.1 and 0.46 ± 0.1,p< 0.01). Compartment modeling and Logan graphical analysis in wistar rats inoculated with hPBMCs indicated that the binding of [18F]FB-IL2v to IL2R was reversible. The volume of distribution (VT) and the non-displaceable binding potential (BPnd) of mutant [18F]FB-IL2v in the implant were approximately 3 times lower than those of wild-type [18F]FB-IL2 (p< 0.01). Pretreatment with wt-IL2 significantly reduced the VTand BPnd of mutant [18F]FB-IL2v in the implant (p< 0.001). This demonstrates that wild-type [18F]FB-IL2 binds stronger to IL2R and has faster kinetics than [18F]FB-IL2v, which makes it less suitable as a therapeutic drug. [18F]FB-IL2v, on the other hand, seems to have better properties for use as a therapeutic drug

Validation of18F-DOPA quality control using UPLC®-MS

Objectives: 6-[18F]Fluoro-L-DOPA (FDOPA) is used to study the presynaptic dopamine synthesis in vivo using PET [1]. Major indications for this tracer are determination of the disease stage in Parkinson as well as detection of various neuroendocrine tumors and their metastases. Prior to set up a Good Manufacturing Practice (GMP) compliant production, the quality control of FDOPA needs to be validated. The radiopharmaceutical needs to meet the specification as described in the European Pharmacopoeia 7.0 Methods: An Ultra Performance Liquid Chromatography UPLC® with an online radioactivity detector was used to measure the concentrations of FDOPA and impurities. For identification of the parent compound and its impurities an UPLC® coupled to a Xevo® G2 QTof mass spectrometer was used. The different components were separated on an Waters ACQUITY UPLC® HSS T3 1.8 μm, 3.0 x 50 mm analytical column. The eluent is 0.05 M sodium dihydrogen phosphate buffer pH 2.5 with an isocratic flow of 0.8 ml/min. Total run time is 3 minutes. Several calibration samples of FDOPA were measured to determine linearity, reproducibility, stability, LOQ, LOD and the resolution. Furthermore the linearity of the Berthold flowstar LB 513 online radioactivity detector was measured. For identification of the parent compound and impurities, a sample was taken directly after the [18F]FDOPA synthesis. The measurements with the mass spectrometer were performed in negative resolution mode with an electrospray source in a range from 50 to 1200 Da. The masses of interest of FDOPA, DOPA, 6-hydroxy- DOPA and FDOPA-quinone (see fig. 1) are respectively 214.0516, 196.0610, 212.0559 and 212.0359 Da. (Figure presented) Results: The analyses using UPLC® in combination with online radioactivity detector were validated. FDOPA and side products DOPA and FDOPA-quinone were detected and identified in the production batches. The concentrations were 1040, 0.1 and 61.5 μg/mL (n = 19), respectively. The samples were measured over a period time of 9 weeks after production and showed a clear increase in the concentration of FDOPA- quinone over time. The DOPA concentration decreasing over this period. In all the samples the concentration of 6-hydroxy- DOPA was below the detection limit and therefore it was not possible to identify this side product with UPLC® -MS. The main impurity was FDOPA-quinone, which is not described in the European Pharmacopoeia 7.0. Conclusions: The quality control of FDOPA using UPLC® -RA was validated for GMP productions. The UPLC® method is reproducible and the retention times of the desired compounds are very short. This is of advantage for rapid releasing the production batch for human application

PET Imaging with S-[C-11]Methyl-L-Cysteine and L-[Methyl-C-11]Methionine in Rat Models of Glioma, Glioma Radiotherapy, and Neuroinflammation

PURPOSE: S-[(11)C]-methyl-L-cysteine ([(11)C]MCYS) has been claimed to offer higher tumor selectivity than L-[methyl- (11)C]methionine ([(11)C]MET). We examined this claim in animal models. PROCEDURES: Rats with implanted untreated (n = 10) or irradiated (n = 7, 1 × 25 Gy, on day 8) orthotopic gliomas were scanned after 6, 9, and 12 days, using positron emission tomography. Rats with striatal injections of saline (n = 9) or bacterial lipopolysaccharide (n = 9) were scanned after 3 days. RESULTS: Uptake of the two tracers in untreated gliomas was similar. [(11)C]MCYS was not accumulated in salivary glands, nasal epithelium, and healing wounds, in contrast to [(11)C]MET, but showed 40 % higher accumulation in the healthy brain. Both tracers showed a reduced tumor uptake 4 days after irradiation and minor accumulation in inflamed striatum. [(11)C]MCYS indicated higher lesion volumes than [(11)C]MET (untreated tumor + 47 %; irradiated tumor up to + 500 %; LPS-inflamed striatum + 240 %). CONCLUSIONS: [(11)C]MCYS was less accumulated in some non-tumor tissues than [(11)C]MET, but showed lower tumor-to-brain contrast

Development of [18F]-labeled pyrazolo[4,3-e]-1,2,4- triazolo[1,5-c]pyrimidine (SCH442416) analogs for the imaging of cerebral adenosine A2A receptors with positron emission tomography

Cerebral adenosine A2A receptors (A2ARs) are attractive therapeutic targets for the treatment of neurodegenerative and psychiatric disorders. We developed high affinity and selective compound 8 (SCH442416) analogs as in vivo probes for A2ARs using PET. We observed the A2AR-mediated accumulation of [18F]fluoropropyl ([18F]-10b) and [18F]fluoroethyl ([18F]-10a) derivatives of 8 in the brain. The striatum was clearly visualized in PET and in vitro autoradiography images of control animals and was no longer visible after pretreatment with the A2AR subtype-selective antagonist KW6002. In vitro and in vivo metabolite analyses indicated the presence of hydrophilic (radio)metabolite(s), which are not expected to cross the blood-brain-barrier. [18F]-10b and [18F]-10a showed comparable striatum-to- cerebellum ratios (4.6 at 25 and 37 min post injection, respectively) and reversible binding in rat brains. We concluded that these compounds performed equally well, but their kinetics were slightly different. These molecules are potential tools for mapping cerebral A2ARs with PET.status: publishe

Synthesis and preclinical evaluation of 2-(2-furanyl)-7-[2-[4-[4-(2-[11C]methoxyethoxy)phenyl]-1-piperazinyl]ethyl]7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine-5-amine ([11C]Preladenant) as a PET tracer for the imaging of cerebral adenosine A2A receptors

2-(2-Furanyl)-7-[2-[4-[4-(2-[C-11]methoxyethoxy)phenyl]-1-piperazinyl]ethyl]7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine-5-amine [(11)C]-3 ([C-11]Preladenant) was developed for mapping cerebral adenosine A2A receptors (A2ARs) with PET. The tracer was synthesized in high specific activity and purity. Tissue distribution was studied by PET imaging, ex vivo biodistribution (BD), and in vitro autoradiography (ARG) experiments. Regional brain uptake of [C-11]-3 was consistent with known A(2A)Rs distribution, with highest uptake in striatum. The results indicate that [C-11]-3 has favorable brain kinetics and exhibits suitable characteristics as an A(2A)R PET tracer

In Vivo Biodistribution of No-Carrier-Added 6-F-18-Fluoro-3, 4-Dihydroxy-L-Phenylalanine (F-18-DOPA), Produced by a New Nucleophilic Substitution Approach, Compared with Carrier-Added F-18-DOPA, Prepared by Conventional Electrophilic Substitution

A novel synthetic approach to 6-F-18-fluoro-3,4-dihydroxy-L-phenylalanine (F-18-DOPA), involving the nucleophilic substitution of a diaryliodonium salt precursor with non-carrier-added F-18-fluoride, yielded a product with a specific activity that was 3 orders of magnitude higher than the product of the conventional synthesis method, involving an electrophilic substitution of a trialkylstannane precursor with F-18(2). We performed a direct comparison of high-and lowspecific-activity F-18-DOPA in a neuroendocrine tumor model to determine whether this difference in specific activity has implications for the biologic behavior and imaging properties of F-18-DOPA. Methods: F-18-DOPA was produced via the novel synthesis method, yielding F-18-DOPA-H with a high specific activity (35,050 +/- 4,000 GBq/mmol). This product was compared in several experiments with conventional F-18-DOPA-L with a low specific activity (11 +/- 2 GBq/mmol). In vitro accumulation experiments with the human pancreatic neuroendocrine tumor cell line BON-1 were performed at both 0 degrees C and 37 degrees C and at 37 degrees C in the presence of pharmacologic inhibitors of proteins involved in the uptake mechanism of F-18-DOPA. Small-animal PET experiments were performed in athymic nude mice bearing a BON-1 tumor xenograft. Results: At 37 degrees C, the uptake of both F-18-DOPA-H and F-18-DOPA-L did not differ significantly during a 60-min accumulation experiment in BON-1 cells. At 0 degrees C, the uptake of F-18-DOPA-L was significantly decreased, whereas the lower temperature did not alter the uptake of F-18-DOPA-H. The pharmacologic inhibitors carbidopa and tetrabenazine also revealed differential effects between the 2 types of F-18-DOPA in the 60-min accumulation experiment. The small-animal PET experiments did not show any significant differences in distribution and metabolism of F-18-DOPA-H and F-18-DOPA-L in carbidopapretreated mice. Conclusion: The advantages of the novel synthesis of F-18-DOPA, which relies on nucleophilic fluorination of a diaryliodonium salt precursor, lie in the simplicity of the synthesis method, compared with the conventional, electrophilic approach and in the reduced mass of administered, pharmacologically active F-19-DOPA. F-18-DOPA-H demonstrated comparable imaging properties in an in vivo model for neuroendocrine tumors, despite the fact that the injected mass of material was 3 orders of magnitude less than F-18-DOPA-L