Synthesis and pharmacological evaluation of [18F]PBR316: a novel PET ligand targeting the translocator protein 18 kDa (TSPO) with low binding sensitivity to human single nucleotide polymorphism rs6971

Abstract

Radiopharmaceuticals that target the translocator protein 18 kDa (TSPO) have been investigated with positron emission tomography (PET) to study neuroinflammation, neurodegeneration and cancer. We have developed the novel, achiral, 2-phenylimidazo[1,2-a]pyridine, PBR316 that targets the translocator protein 18 kDa (TSPO) that addresses some of the limitations inherent in current TSPO ligands; namely specificity in binding, blood brain barrier permeability, metabolism and insensitivity to TSPO binding in subjects as a result of rs6971 polymorphism. PBR316 has high nanomolar affinity (4.7-6.0 nM) for the TSPO, >5000 nM for the central benzodiazepine receptor (CBR) and low sensitivity to rs6971 polymorphism with a low affinity binders (LABs) to high affinity binders (HABs) ratio of 1.5. [¹⁸F]PBR316 was prepared in 20 ± 5% radiochemical yield, >99% radiochemical purity and a molar activity of 160-400 GBq μmol^-1. Biodistribution in rats showed high uptake of [¹⁸F]PBR316 in organs known to express TSPO such as heart (3.9%) and adrenal glands (7.5% ID per g) at 1 h. [¹⁸F]PBR316 entered the brain and accumulated in TSPO-expressing regions with an olfactory bulb to brain ratio of 3 at 15 min and 7 at 4 h. Radioactivity was blocked by PK11195 and Ro 5-4864 but not Flumazenil. Metabolite analysis showed that radioactivity in adrenal glands and the brain was predominantly due to the intact radiotracer. PET-CT studies in mouse-bearing prostate tumour xenografts indicated biodistribution similar to rats with radioactivity in the tumour increasing with time. [¹⁸F]PBR316 shows in vitro binding that is insensitive to human polymorphism and has specific and selective in vivo binding to the TSPO. [¹⁸F]PBR316 is suitable for further biological and clinical studies.

Fig. 1. Chemical structure of imidazopyridine-based TSPO ligands – [¹⁸F]PBR111 and [¹⁸F]PBR102.

Fig. 2. General structure of a 2-phenyl imidazopyridine (a) and the oxoacetamide derivative PBR316.

Scheme 1. Synthesis of PBR316 and its tosyl precursor 5. a) Br₂, AlCl₃, ethyl acetate. b) 2-Amino-5-chloropyridine, NaHCO₃, EtOH. c) COCl₂, DIPEA, CH₂Cl₂, NH(Me₃)₂, pyridine. d) K₂CO₃, MeOH. e) TsCl, TMHDA, or TsCl, K₂CO₃, dry grind method. f) PFBSF, NEt₃·3HF, DIPEA, CH₃CN.

Scheme 2. Radiosynthesis of [¹⁸F]PBR316. a) [¹⁸F]fluoride, K₂CO₃, K₂₂₂, CH₃CN.

Fig. 3. HPLC analysis of formulated [¹⁸F]PBR316 and PBR316 standard (a) radio-HPLC chromatogram of [¹⁸F]PBR316 (b) HPLC UV-chromatogram of [¹⁸F]PBR316 (c) HPLC chromatogram of PBR316 standard.

Fig. 4. Competition assays with [³H]PK11195 in the presence of increasing concentrations of PBR316 using human platelets previously characterised as HABs or LABs. Each data point represents mean ± SD, n = 3.

Fig. 5. Effect of competing drugs (1 mg kg⁻¹; PK11195, PBR316, Ro 5-4864, and flumazenil) on [¹⁸F]PBR316 uptake in CNS of rat at 1 h p.i. Results are mean ± SD, unit is % injected dose per g tissue, n = 4 controls and n = 3–4 treated animals (*p < 0.01).

Fig. 6. Effect of competing drugs (1 mg kg⁻¹; PK11195, PBR316, Ro 5-4864, and flumazenil) on [¹⁸F]PBR316 uptake in peripheral organs and skull of rat at 1 h p.i. results are mean ± SD, unit is % injected dose per g tissue, n = 4 controls and n = 3–4 treated animals. (*p < 0.01).

Fig. 7. Percentage of un-metabolized [¹⁸F]PBR316 in tissues and plasma as measured using solid phase extraction.

Fig. 8. Time course distribution of radioactivity in blood and in red and white blood cells as measured directly in haematocrit tubes by phosphorimaging. The percentage of un-metabolized [¹⁸F]PBR316 was determined by SPE collecting each fraction of blood components from the haematocrit tubes. Results are presented as the % of injected dose (% ID) in 1 mL of rat blood after injection of 30–40 MBq of radiotracer. Two hours after injection, more than 50% of unchanged [¹⁸F]PBR316 is in WBC. Total radioactivity is reported by full lines, unchanged [¹⁸F]PBR316 in dotted lines.

Fig. 9. Distribution of radioactivity in blood components 15 min after [¹⁸F]PBR316 (30–40 MBq) injection in rats as determined using haematocrit and phosphorimaging techniques. Picture of the capillary tube with blood components after centrifugation: (a) image of the capillary tube showing blood components; (b) autoradiographic image of the radioactivity distribution in blood components; (c) autoradiographic image showing the radioactivity distribution in the blood after in vitro displacement with PBR316 (60 nmol); (d) distribution profile of radioactivity in blood components as a function of tube length. Values represent the percentage of radioactivity in 3 mm zone to the total radioactivity in the tube. The major part of the radioactivity (76%) was found in the WBC zone and was displaced by PBR316.

Fig. 10. Representative fused PET–CT data at 30 min post-injection of [¹⁸F]PBR316 (10 MBq) in a mouse with a subcutaneous PC-3M-Luc-C6 xenograft (a). Time course of tumour uptake measured in vivo with PET and ex vivo with γ-counter over 240 min (b).

Fig. 11. Time course of radioactivity measured with PET in the heart and tumour of a mouse with a subcutaneous PC-3M-Luc-C6 xenograft after intravenous injection of [¹⁸F]PBR316 (3–20 MBq) and displacement with 1 mg kg⁻¹ PK11195 intravenously injected 30 min after the radiotracer.