GMP Compliant Synthesis of [18F]Canagliflozin, a Novel PET Tracer for the Sodium-Glucose Cotransporter 2.

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 [18F]canagliflozin was developed via a Cu-mediated 18F-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 [18F]canagliflozin with a yield of 0.5-3% (n = 4) and a purity of >95%. Autoradiography showed [18F]canagliflozin binding in human kidney sections containing SGLT2. Since [18F]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 IL2αßγ receptor, but only to its ß 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 VT and 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.

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

PURPOSE: S-[11C]-methyl-L-cysteine ([11C]MCYS) has been claimed to offer higher tumor selectivity than L-[methyl- 11C]methionine ([11C]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. [11C]MCYS was not accumulated in salivary glands, nasal epithelium, and healing wounds, in contrast to [11C]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. [11C]MCYS indicated higher lesion volumes than [11C]MET (untreated tumor + 47 %; irradiated tumor up to + 500 %; LPS-inflamed striatum + 240 %). CONCLUSIONS: [11C]MCYS was less accumulated in some non-tumor tissues than [11C]MET, but showed lower tumor-to-brain contrast.

Contribution of neuroinflammation to changes in [11C]flumazenil binding in the rat brain: Evaluation of the inflamed pons as reference tissue.

INTRODUCTION: [11C]Flumazenil is a well-known PET tracer for GABAA receptors and is mainly used as an imaging biomarker for neuronal loss. Recently, GABAA receptors on immune cells have been investigated as target for modulation of inflammation. Since neuronal loss is often accompanied by neuroinflammation, PET imaging with [11C]flumazenil is potentially affected by infiltrating immune cells. This may also compromise the validity of using the pons as reference tissue in quantitative pharmacokinetic analysis. This study aims to evaluate whether inflammatory processes in the brain can influence [11C]flumazenil uptake and affect the outcome of pharmacokinetic modeling when the pons is used as reference tissue. METHODS: The herpes simplex encephalitis (HSE) rat model is known to cause neuroinflammation in the brainstem. Dynamic [11C]flumazenil PET scans of 60-min, accompanied by arterial blood sampling and metabolite analysis, were acquired at day 6-7days post-infection of male Wistar rats (HSE, n=5 and control, n=6). Additionally, the GABAA receptor was saturated by injection of unlabeled flumazenil prior to the tracer injection in 4 rats per group. PET data were analyzed by pharmacokinetic modeling. RESULTS: No statistically significant differences were found in the volume of distribution (VT) or non-displaceable binding potential (BPND) between control and HSE rats in any of the brain regions. Pre-saturation with unlabeled flumazenil resulted in a statistically significant reduction in [11C]flumazenil VT in all brain regions. The BPND obtained from SRTM exhibited a good correlation to DVR - 1 values from the two-tissue compartment model, coupled with some level of underestimation. CONCLUSION: Reliable quantification of [11C]flumazenil binding in rats can be obtained by pharmacokinetic analysis using the pons as a pseudo-reference tissue even in the presence of strong acute neuroinflammation.

In vivo biodistribution of no-carrier-added 6-18F-fluoro-3,4-dihydroxy-L-phenylalanine (18F-DOPA), produced by a new nucleophilic substitution approach, compared with carrier-added 18F-DOPA, prepared by conventional electrophilic substitution.

UNLABELLED: A novel synthetic approach to 6-(18)F-fluoro-3,4-dihydroxy-L-phenylalanine ((18)F-DOPA), involving the nucleophilic substitution of a diaryliodonium salt precursor with non-carrier-added (18)F-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 (18)F2. We performed a direct comparison of high- and low-specific-activity (18)F-DOPA in a neuroendocrine tumor model to determine whether this difference in specific activity has implications for the biologic behavior and imaging properties of (18)F-DOPA. METHODS: (18)F-DOPA was produced via the novel synthesis method, yielding (18)F-DOPA-H with a high specific activity (35,050 ± 4,000 GBq/mmol). This product was compared in several experiments with conventional (18)F-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 °C and 37 °C and at 37 °C in the presence of pharmacologic inhibitors of proteins involved in the uptake mechanism of (18)F-DOPA. Small-animal PET experiments were performed in athymic nude mice bearing a BON-1 tumor xenograft. RESULTS: At 37 °C, the uptake of both (18)F-DOPA-H and (18)F-DOPA-L did not differ significantly during a 60-min accumulation experiment in BON-1 cells. At 0 °C, the uptake of (18)F-DOPA-L was significantly decreased, whereas the lower temperature did not alter the uptake of (18)F-DOPA-H. The pharmacologic inhibitors carbidopa and tetrabenazine also revealed differential effects between the 2 types of (18)F-DOPA in the 60-min accumulation experiment. The small-animal PET experiments did not show any significant differences in distribution and metabolism of (18)F-DOPA-H and (18)F-DOPA-L in carbidopa-pretreated mice. CONCLUSION: The advantages of the novel synthesis of (18)F-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 (19)F-DOPA. (18)F-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 (18)F-DOPA-L.

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-[(11)C]methoxyethoxy)phenyl]-1-piperazinyl]ethyl]7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine-5-amine [(11)C]-3 ([(11)C]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 [(11)C]-3 was consistent with known A2ARs distribution, with highest uptake in striatum. The results indicate that [(11)C]-3 has favorable brain kinetics and exhibits suitable characteristics as an A2AR PET tracer.

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.