Development of [¹²³I]IPEB and [¹²³I]IMPEB as SPECT Radioligands for Metabotropic Glutamate Receptor Subtype 5

mGlu₅ play an important role in physiology and pathology to various central nervous system (CNS) diseases. Several positron emission tomography (PET) radiotracers have been developed to explore the role of mGlu₅ in brain disorders. However, there are no single photon emission computed tomography (SPECT) radioligands for mGlu₅. Here we report development of [¹²³I]­IPEB ([¹²³I]<b>1</b>) and [¹²³I]­IMPEB ([¹²³I]<b>2</b>) as mGlu₅ radioligands for SPECT. [¹²³I]<b>1</b> and [¹²³I]<b>2</b> were produced by copper­(I) mediated aromatic halide displacement reactions. The SPECT imaging using mouse models demonstrated that [¹²³I]<b>1</b> readily entered the brain and accumulated specifically in mGlu₅-rich regions of the brain such as striatum and hippocampus. However, in comparison to the corresponding PET tracer [¹⁸F]­FPEB, [¹²³I]<b>1</b> showed faster washout from the brain. The binding ratios of the striatum and the hippocampus compared to the cerebellum for [¹²³I]<b>1</b> and [¹⁸F]­FPEB were similar despite unfavorable pharmacokinetics of [¹²³I]<b>1</b>. Further structural optimization of <b>1</b> may lead to more viable SPECT radiotracers for the imaging of mGlu₅

Radiosynthesis and Evaluation of an ¹⁸F‑Labeled Positron Emission Tomography (PET) Radioligand for Metabotropic Glutamate Receptor Subtype 4 (mGlu₄)

Four 4-phthalimide derivatives of <i>N</i>-(3-chlorophenyl)-2-picolinamide were synthesized as potential ligands for the PET imaging of mGlu₄ in the brain. Of these compounds, <i>N</i>-(3-chloro-4-(4-fluoro-1,3-dioxoisoindolin-2-yl)­phenyl)-2-picolinamide (<b>3</b>, KALB001) exhibited improved binding affinity (IC₅₀ = 5.1 nM) compared with ML128 (<b>1</b>) and was subsequently labeled with ¹⁸F. When finally formulated in 0.1 M citrate buffer (pH 4) with 10% ethanol, the specific activity of [¹⁸F]<b>3</b> at the end of synthesis (EOS) was 233.5 ± 177.8 GBq/μmol (<i>n</i> = 4). The radiochemical yield of [¹⁸F]<b>3</b> was 16.4 ± 4.8% (<i>n</i> = 4), and the purity was over 98%. In vivo imaging studies in a monkey showed that the radiotracer quickly penetrated the brain with the highest accumulation in the brain areas known to express mGlu₄. Despite some unfavorable radiotracer properties like fast washout in rodent studies, [¹⁸F]<b>3</b> is the first ¹⁸F-labeled mGlu₄ radioligand, which can be further modified to improve pharmacokinetics and brain penetrability for future human studies

Astrocytic marker was decreased in the brain of prenatally saline-exposed offspring, but not in the offspring prenatally exposed to LPS, following pharmacological activation of mGluR5.

<p>A prenatal administration of LPS did not change the brain expression level of iba1, CD68, GFAP or mGluR5 in 4-month-old mice (A). Postnatal CDPPB treatment reduced the GFAP level, without effect on iba1, CD68 or mGluR5 in the mice prenatally exposed to saline solution. The quantified molecular markers did not change in the brain of mice treated with MTEP (B). No change in iba1, CD68, GFAP or mGluR5 was observed in the brain of the mice prenatally exposed to LPS (C). Values are expressed as mean ± SEM. Abbreviations: MTEP, 3-((2-methyl-4-thiazolyl)ethynyl)pyridine; CDPPB, 3-cyano-<i>N</i>-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide; CD68, cluster of differentiation 68); iba1, ionized calcium binding adaptor molecule-1; GFAP, glial fibrillary acidic protein; mGluR5, metabotropic glutamate receptor subtype 5; ROD, relative optical density. **p < 0.01 >></p

Effect of aging and different prenatal treatments on mGluR5 expression investigated with PET imaging using [¹⁸F]FPEB.

<p>PET imaging revealed a significant increase of [¹⁸F]FPEB binding during maturation (from PnD37-39 to PnD 117–119) in the striatum and the whole brain of the mice prenatally exposed to saline (A), while in the LPS-exposed offspring the [¹⁸F]FPEB binding increased in the cortex, hippocampus, striatum and the whole brain during the same maturation period (B). During the adolescence (PnD37-39) we observed a lower binding of [¹⁸F]FPEB in the striatum and the whole brain of the LPS-exposed offspring compared to the saline-treated offspring (C). However, [¹⁸F]FPEB binding was similar in both groups at PnD117-119 (D). (E) Coronal slices of hippocampal and striatal level of [¹⁸F]FPEB in offspring prenatally exposed to saline or LPS at PnD 37–39 and PnD 117–119. Values are expressed as mean ± SEM. Abbreviations: [¹⁸F]fluoro-5-(2-pyridinylethynyl)benzonitrile; Ctx, cortex; Hip, hippocampus; Hth, hypothalamus; OB, olfactory bulb; PnD, postnatal day; Str, striatum; W, whole brain. *p < 0.05, **p < 0.01 >></p

Postnatal CDPPB treatment decreased [¹¹C]PBR accumulation in saline-exposed offspring, but not in offspring prenatally exposed to LPS.

<p>Postnatal CDPPB treatment decreased the accumulation of [¹¹C]PBR in the olfactory bulb, cerebellum, hippocampus, striatum, hypothalamus and the whole brain of the offspring prenatally exposed to saline solution (A). However, [¹¹C]PBR accumulation did not change in any quantified brain region of the LPS-exposed offspring (B). Axial PET images of [¹¹C]PBR accumulation at the midbrain level illustrate the decreased accumulation after CDPPB treatment in the prenatally saline-exposed offspring while there is no significant change in the brain of CDPPB treated mice prenatally exposed to LPS (C). Values are expressed as mean ± SEM. Abbreviations: CDPPB, 3-cyano-<i>N</i>-(1,3-diphenyl-1<i>H</i>-pyrazol-5-yl)benzamide; [¹¹C] PBR28, peripheral benzodiazepine receptor 28; Ctx, cortex; Crbl, cerebellum; Hip, hippocampus; Hth, hypothalamus; OB, olfactory bulb; PnD, postnatal day; Str, striatum; W, whole brain. *p < 0.05, **p < 0.01 >></p

Effects of postnatal CDPPB treatment on [¹⁸F]FPEB binding potential.

<p>CDPPB treatment did not change the [¹⁸F]FPEB binding potential in the quantified brain region of the offspring prenatally exposed to saline solution (A). However, the LPS-exposed offspring had a lower [¹⁸F]FPEB accumulation in the cortex and hippocampus following CDPPB treatment (B). (C) Axial view of a representative mouse from each group. Values are expressed as mean ± SEM. Abbreviations: CDPPB, 3-cyano-<i>N</i>-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide; [¹⁸F]FPEB, [18F]fluoro-5-(2-pyridinylethynyl)benzonitrile; Ctx, cortex; Hip, hippocampus; Hth, hypothalamus; OB, olfactory bulb; PnD, postnatal day; Str, striatum; W, whole brain. *p < 0.05, **p < 0.01. Statistical analyses were performed using one-sample t test (all comparisons of panel A; OB, Hip, Str, Hth and W of panel B) or Wilcoxon signed-rank test (Ctx of panel B). The number of animals was 10–11 for the saline group and 12 for the LPS-exposed group. >></p

Time lines of the experiments.

<p>Pregnant mice received an injection of 120 μg/kg of LPS or an equivalent volume of saline between GD15 to GD17. Expression of mGluR5 (PnD37-PnD39) and PBR (PnD42-PnD44) in the brain of the offspring prenatally exposed to LPS or saline were evaluated by PET imaging. Animals of both groups (LPS or saline) were then exposed to treatment during 5 weeks (from PnD56 to PnD91). Three types of treatments were tested: CDPPB (10 mg/kg; mGluR5 agonist), MTEP (3 mg/kg; mGluR5 antagonist) and control (solvent alone). Finally, the <i>in vivo</i> expression of mGluR5 (PnD117-119) and PBR (PnD125-127) were re-evaluated after the treatments and the animals were sacrificed a few days after the last imaging session (PnD128-130) for post-mortem analysis. Abbreviations: GD, gestational day; [¹¹C]PBR28, peripheral benzodiazepine receptor 28; [¹⁸F]FPEB, [¹⁸F]fluoro-5-(2-pyridinylethynyl)benzonitrile; PnD, postnatal day.>></p